Thermally stable glucose limiting membrane for glucose sensors

ABSTRACT

Embodiments of the invention provide compositions useful in analyte sensors as well as methods for making and using such compositions and sensors. In typical embodiments of the invention, the sensor is a glucose sensor comprising an analyte modulating membrane formed from a polymeric reaction mixture formed to include limiting amounts of catalyst and/or polycarbonate compounds so as to provide such membranes with improved material properties such as enhanced thermal and hydrolytic stability.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application which claims the benefitunder 35 U.S.C. § 120 and § 121 of U.S. patent application Ser. No.15/981,681, and now U.S. Pat. No. 11,134,872 filed May 16, 2018, whichclaims priority under Section 120 from U.S. patent application Ser. No.15/612,759, and now U.S. Pat. No. 11,179,078 filed Jun. 2, 2017,entitled “POLYCARBONATE UREA/URETHANE POLYMERS FOR USE WITH ANALYTESENSORS”, which claims the benefit under 35 U.S.C. Section 119(e) ofU.S. Provisional Application Ser. No. 62/346,301, filed on Jun. 6, 2016.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates to biosensors such as glucose sensors used in themanagement of diabetes and materials for making such sensors, forexample polymeric compositions useful for biosensor membranes.

2. Description of Related Art

Analyte sensors such as biosensors include devices that use biologicalelements to convert a chemical analyte in a matrix into a detectablesignal. There are many types of biosensors used to detect wide varietyof analytes. Perhaps the most studied type of biosensor is theamperometric glucose sensor, an apparatus commonly used to monitorglucose levels in individuals with diabetes.

A typical glucose sensor works according to the following chemicalreactions:

The glucose oxidase is used to catalyze the reaction between glucose andoxygen to yield gluconic acid and hydrogen peroxide as shown inequation 1. The H₂O₂ reacts electrochemically as shown in equation 2,and the current is measured by a potentiostat. The stoichiometry of thereaction provides challenges to developing in vivo sensors. Inparticular, for optimal sensor performance, sensor signal output shouldbe determined only by the analyte of interest (glucose), and not by anyco-substrates (O₂) or kinetically controlled parameters such asdiffusion. If oxygen and glucose are present in equimolarconcentrations, then the H₂O₂ is stoichiometrically related to theamount of glucose that reacts at the enzyme; and the associated currentthat generates the sensor signal is proportional to the amount ofglucose that reacts with the enzyme. If, however, there is insufficientoxygen for all of the glucose to react with the enzyme, then the currentwill be proportional to the oxygen concentration, not the glucoseconcentration. Consequently, for the sensor to provide a signal thatdepends solely on the concentrations of glucose, glucose must be thelimiting reagent, i. e. the O₂ concentration must be in excess for allpotential glucose concentrations. A problem with using such glucosesensors in vivo, however, is that the oxygen concentration where thesensor is implanted in vivo is low relative to glucose, phenomena whichcan compromise the accuracy of sensor readings.

There are a number of approaches to solving the oxygen deficit problem.One is to use a homogenous polymer membrane with hydrophobic andhydrophilic regions that control oxygen and glucose permeability. Forexample, Van Antwerp et al. developed linear polyurea membranescomprising polyethylene glycol and silicone hydrophobic components thatallow for a high oxygen permeability in combination with hydrophiliccomponent that allow for a limited glucose permeability (see e.g. U.S.Pat. Nos. 5,777,060, 5,882,494 and 6,642,015). While having a number ofuseful and desirable characteristics, such polymeric compositions canexperience some degradation over time under high temperature and highhumidity conditions. In view of this, there is a need in the art formore robust polymeric membrane compositions that can, for example, beused to address the oxygen deficit problem that is observed in glucosesensors that incorporate glucose oxidase.

SUMMARY OF THE INVENTION

Embodiments of the invention provide compositions useful in analytesensors as well as methods for making and using such compositions andsensors. In typical embodiments of the invention, the sensor is aglucose sensor comprising an analyte modulating membrane formed from apolymeric reaction mixture that includes limiting amounts of catalyst soas to provide such membranes with improved material properties such asenhanced thermal and hydrolytic stability. As disclosed herein, whenthese polymer compositions are used to form the analyte limitingmembranes in glucose sensors, the resultant sensors exhibit enhanced thelong term stability profiles as compared to conventional polymercompositions formed from reaction mixtures that use conventional amountsof catalyst.

The invention disclosed herein has a number of embodiments. Oneembodiment of the invention is a method of increasing the thermalstability of a biocompatible membrane formed by a reaction mixturecomprising a diisocyanate, a hydrophilic polymer comprising ahydrophilic diol or hydrophilic diamine, a siloxane having an amino,hydroxyl or carboxylic acid functional group at a terminus; and acatalyst. In this methodology, the reaction mixture is formed so thatthe catalyst is present in the reaction mixture in amounts less than0.2% of reaction mixture components (e.g. 0.1%), thereby increasing thethermal stability of the biocompatible membrane as compared to acomparable membrane formed from a reaction mixture where the catalyst ispresent in the formulation in amounts greater than or equal to 0.2% ofthe reaction mixture. Optionally the reaction mixture further comprisesadditional components such as a polycarbonate diol.

Another embodiment of the invention is an amperometric analyte sensorcomprising a base layer, a conductive layer disposed on the base layerand comprising a working electrode, an analyte sensing layer disposed onthe conductive layer, and an analyte modulating layer disposed on theanalyte sensing layer. In this embodiment, the analyte modulating layeris formed by a reaction mixture comprising a diisocyanate, a hydrophilicpolymer comprising a hydrophilic diol or hydrophilic diamine, a siloxanehaving an amino, hydroxyl or carboxylic acid functional group at aterminus, and a catalyst. In this embodiment, the amount of catalystpresent in the reaction mixture in amounts less than 0.2% of reactionmixture components so that the analyte modulating layer exhibits agreater thermal stability than a comparable analyte modulating layerformed from a reaction mixture where the catalyst is present in theformulation in amounts greater than or equal to 0.2% of the reactionmixture.

Yet another embodiment of the invention is a method of making an analytesensor for implantation within a mammal. This methodological embodimentcomprises the steps of providing a base layer, forming a conductivelayer on the base layer, wherein the conductive layer includes a workingelectrode, forming an analyte sensing layer on the conductive layer,wherein the analyte sensing layer includes an oxidoreductase, and thenforming an analyte modulating layer on the analyte sensing layer. Inthis embodiment, the analyte modulating layer is formed by a reactionmixture comprising a diisocyanate, a hydrophilic polymer comprising ahydrophilic diol or hydrophilic diamine, a siloxane having an amino,hydroxyl or carboxylic acid functional group at a terminus; and acatalyst present in the reaction mixture in amounts less than 0.2% (e.g.0.1%) of reaction mixture components so that the reaction mixtureexhibits a greater thermal stability than a comparable analytemodulating layer formed from a reaction mixture where the catalyst ispresent in the formulation in amounts greater than or equal to 0.2% ofthe reaction mixture. Optionally the reaction mixture further comprisesadditional components such as a polycarbonate diol.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating some embodiments ofthe present invention are given by way of illustration and notlimitation. Many changes and modifications within the scope of thepresent invention may be made without departing from the spirit thereof,and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE FIGURES

Referring now to the drawings in which like reference numbers representcorresponding parts throughout:

FIG. 1 provides a diagrammatic view of one embodiment of an amperometricanalyte sensor having a plurality of layered materials/elements, inaccordance with one or more embodiments of the invention;

FIGS. 2A-C show the chemical structures of raw materials used in thepolycarbonate urea glucose limiting membrane (GLM), in accordance withone or more embodiments of the invention. FIG. 2A shows the chemicalstructures of PDMS and Jeffamine. FIG. 2B shows the chemical structuresof 4,4′-Methylenebis(cyclohexyl isocyanate) or HMDI and4,4′-Methylenebis(phenyl isocyanate) or MDI. FIG. 2C shows the chemicalstructure of polycarbonate diols;

FIG. 3 illustrates a GLM synthesis reaction, in accordance with one ormore embodiments of the invention;

FIGS. 4A-B show a comparison of morphology of various counter andworking electrodes after testing, in accordance with one or moreembodiments of the invention. FIG. 4A shows bubbles (or craters)generated at the counter electrode after usage. Bubbles formation at thecounter electrode may trigger delamination or unwanted biologicalresponses (due to texture change or rough surface). FIG. 4B shows thatthe MDI_polycarnobate_GLM can enhance the GLM adhesion, so that bubbles(or craters) are not generated at the counter electrode after usage;

FIG. 5 shows an in vitro SITS data comparison between standard 2×GLM andPCU_GLM (polycarbonate urea glucose limiting membrane), in accordancewith one or more embodiments of the invention;

FIG. 6 shows E3 sensor morphology after 7 days SITS testing for standard2×GLM coated sensors and PCU_GLM coated sensors, in accordance with oneor more embodiments of the invention;

FIGS. 7A-B provide graphs of data from thermal degradation studies forvarious formulations and the compositions of the formulations. FIG. 7Aprovides the results from a thermal degradation study at 45° C.comparing the degradation of polymeric materials (as observed by adecrease in molecular weight) useful as biocompatible membranes (e.g.analyte modulating layers in glucose sensors), while FIG. 7B providesthe results from a similar thermal degradation study at 60° C. In FIGS.7A and 7B, the “control” material is formed from a polymeric reactionmixture where the catalyst is present in the formulation in amountsgreater than or equal to 0.2% of the polymeric reaction mixture and the“new” material is formed from a polymeric reaction mixture where thecatalyst is present in the formulation in amounts less than 0.2% (inthis case 0.1%) of the reaction mixture.

FIGS. 8A-E show results from thermal degradation studies for variousformulations and the compositions of the formulations, in accordancewith one or more embodiments of the invention. FIG. 8A shows the resultsfrom a thermal degradation study. FIGS. 8B-D show the compositions ofvarious formulations. FIG. 8E provides data showing that glucosepermeability (Pg) was not reduced after baking for polycarbonate_GLM;

FIG. 9 shows a summary of the results from thermal/hydrolysis studies ofvarious sample formulations, in accordance with one or more embodimentsof the invention. The thermal degradation test results demonstrate thatMDI and polycarbonates chains can help (slow down) the GLM degradationprocess.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted. A number of termsare defined below. All publications mentioned herein are incorporatedherein by reference to disclose and describe the methods and/ormaterials in connection with which the publications are cited.Publications cited herein are cited for their disclosure prior to thefiling date of the present application. Nothing here is to be construedas an admission that the inventors are not entitled to antedate thepublications by virtue of an earlier priority date or prior date ofinvention. Further the actual publication dates may be different fromthose shown and require independent verification.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anoxidoreductase” includes a plurality of such oxidoreductases andequivalents thereof known to those skilled in the art, and so forth. Allnumbers recited in the specification and associated claims that refer tovalues that can be numerically characterized with a value other than awhole number (e.g. “50 mol %”) are understood to be modified by the term“about”.

The term “analyte” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to a substanceor chemical constituent in a fluid such as a biological fluid (forexample, blood, interstitial fluid, cerebral spinal fluid, lymph fluidor urine) that can be analyzed. Analytes can include naturally occurringsubstances, artificial substances, metabolites, and/or reactionproducts. In some embodiments, the analyte for measurement by thesensing regions, devices, and methods is glucose. However, otheranalytes are contemplated as well, including but not limited to,lactate. Salts, sugars, proteins fats, vitamins and hormones naturallyoccurring in blood or interstitial fluids can constitute analytes incertain embodiments. The analyte can be naturally present in thebiological fluid or endogenous; for example, a metabolic product, ahormone, an antigen, an antibody, and the like. Alternatively, theanalyte can be introduced into the body or exogenous, for example, acontrast agent for imaging, a radioisotope, a chemical agent, afluorocarbon-based synthetic blood, or a drug or pharmaceuticalcomposition, including but not limited to insulin. The metabolicproducts of drugs and pharmaceutical compositions are also contemplatedanalytes.

The term “sensor,” as used herein, is a broad term and is used in itsordinary sense, including, without limitation, the portion or portionsof an analyte-monitoring device that detects an analyte. In oneembodiment, the sensor includes an electrochemical cell that has aworking electrode, a reference electrode, and optionally a counterelectrode passing through and secured within the sensor body forming anelectrochemically reactive surface at one location on the body, anelectronic connection at another location on the body, and a membranesystem affixed to the body and covering the electrochemically reactivesurface. During general operation of the sensor, a biological sample(for example, blood or interstitial fluid), or a portion thereof,contacts (directly or after passage through one or more membranes ordomains) an enzyme (for example, glucose oxidase); the reaction of thebiological sample (or portion thereof) results in the formation ofreaction products that allow a determination of the analyte level in thebiological sample.

As discussed in detail below, embodiments of the invention relate to theuse of an electrochemical sensor that exhibits a novel constellation ofmaterial and functional elements. Such sensors incorporate new polymericcompositions in order to form robust analyte modulating membranes, oneshaving a unique set of technically desirable material properties such asincreased thermal stability. The electrochemical sensors of theinvention are designed to measure a concentration of an analyte ofinterest (e.g. glucose) or a substance indicative of the concentrationor presence of the analyte in fluid. In some embodiments, the sensor isa continuous device, for example a subcutaneous, transdermal, orintravascular device. In some embodiments, the device can analyze aplurality of intermittent blood samples. The sensor embodimentsdisclosed herein can use any known method, including invasive, minimallyinvasive, and non-invasive sensing techniques, to provide an outputsignal indicative of the concentration of the analyte of interest.Typically, the sensor is of the type that senses a product or reactantof an enzymatic reaction between an analyte and an enzyme in thepresence of oxygen as a measure of the analyte in vivo or in vitro. Suchsensors comprise a polymeric membrane surrounding the enzyme throughwhich an analyte migrates prior to reacting with the enzyme. The productis then measured using electrochemical methods and thus the output of anelectrode system functions as a measure of the analyte. In someembodiments, the sensor can use an amperometric, coulometric,conductimetric, and/or potentiometric technique for measuring theanalyte.

Analyte modulating compositions such as those useful as glucose limitingmembranes in amperometric glucose sensors include polymeric compositionsformed from biocompatible polymeric polyurea materials (see, e.g., thecontexts of which re incorporated by reference). Such compositions canexhibit stable glucose and oxygen permeabilities, low protein adsorptionrates, and biocompatibility. However, due to the content of PEG chains,it suffered some degradation issue under high temperature and/or highhumidity conditions. As disclosed in detail below, we have discoveredthat certain carbonate and aromatic isocyanate compounds can be added toa polymerization reaction so as to replace some portions of PDMS andHMDI polymeric chain elements. Both compounds have been discovered toincrease the thermal and hydrolysis resistance of these polymers underhigh temperature and high humidity conditions. In addition, theirchemical structures provide evidence that such compositions have a verygood e-beam resistance. The carbonate materials useful in embodiments ofthe invention include, but are not limited to, polycarbonate diols (e.g.butanediol or hexanediol or similar compounds). In the illustrativeembodiments of the invention, their Mw is from 500 to 2000 Daltons. Thearomatic isocyanate materials useful in embodiments of the inventioninclude, but not limited to, MDI or similar compounds.

In addition to limiting amounts of catalyst in the polymeric reactionmixture, the addition of MDI can improve the thermal and e-beamresistance of polymeric compositions used as analyte modulating (e.g.glucose limiting) compositions through its benzene ring structure. Thebenzene ring also serves as a good free-radical scavenger to preventoxidation of polymeric constituents. The polycarbonate diol can providebetter thermal and hydrolysis resistances through its carbonatestructure (vs. ether or ester chains). The addition of polycarbonatesegment in the polymer backbone can prevent the unwanted deformation ofa layer of a polymer composition that is disposed on an electrode of anamperometric glucose sensor. Both gas and water are generated on acounter electrode between analyte sensing layers (e.g. ones comprised ofan enzyme such as GOX) and analyte modulating (e.g. Glucose LimitingMembrane) layers, which can cause sensor failure (signal drifting) aftera long usage. In this context, the polycarbonate segments in the GLMbackbone can prevent/reduce the chain rotation of PDMS in the GLM film,so the glucose permeability (Pg) of GLM will not be gradually reducedover time due to the hydrophilic chains (Jeffamine or PEG) waswrapped/entrapped by the hydrophobic PDMS chains, especially for the lowPg GLM cases. In certain embodiments, in order to make a homogeneousurethane/urea copolymer, the synthesis will involve 3 raw materialinjections after different timings. The raw materials were injected at4-2-4 ratio at time=0, 4, 12 hours, respectively. The addition ofpolycarbonate chains in the GLM can prevent the Pg change/reduction dueto the PDMS chain rotation/tangling over time, especially for low Pg GLMfilms. In order to reduce thermal/radiation/oxidation degradation, thedesired MDI content in the final polymer can be from 2% to 25%. In orderto prevent the film deformation or Pg reduction due to silicone chainrotation over time, the desired polycarbonate content in the finalpolymer can be from 8% to 30%. Polycarbonate GLM showed good adhesionwith AP, no more craters (bubbles) formed after testing

Embodiments of the invention disclosed herein provide sensors of thetype used, for example, in subcutaneous or transcutaneous monitoring ofblood glucose levels in a diabetic patient. A variety of implantable,electrochemical biosensors have been developed for the treatment ofdiabetes and other life-threatening diseases. Many existing sensordesigns use some form of immobilized enzyme to achieve theirbio-specificity. Embodiments of the invention described herein can beadapted and implemented with a wide variety of known electrochemicalsensors, including for example, U.S. Patent Application No. 20050115832,U.S. Pat. Nos. 6,001,067, 6,702,857, 6,212,416, 6,119,028, 6,400,974,6,595,919, 6,141,573, 6,122,536, 6,512,939 5,605,152, 4,431,004,4,703,756, 6,514,718, 5,985,129, 5,390,691, 5,391, 250, 5,482,473,5,299,571, 5,568,806, 5,494,562, 6,120,676, 6,542,765 as well as PCTInternational Publication Numbers WO 01/58348, WO 04/021877, WO03/034902, WO 03/035117, WO 03/035891, WO 03/023388, WO 03/022128, WO03/022352, WO 03/023708, WO 03/036255, WO03/036310 WO 08/042625, and WO03/074107, and European Patent Application EP 1153571, the contents ofeach of which are incorporated herein by reference.

As discussed in detail below, embodiments of the invention disclosedherein provide sensor elements having enhanced material propertiesand/or architectural configurations and sensor systems (e.g. thosecomprising a sensor and associated electronic components such as amonitor, a processor and the like) constructed to include such elements.The disclosure further provides methods for making and using suchsensors and/or architectural configurations. While some embodiments ofthe invention pertain to glucose and/or lactate sensors, a variety ofthe elements disclosed herein (e.g. analyte modulating membranes madefrom polycarbonate polymeric compositions) can be adapted for use withany one of the wide variety of sensors known in the art. The analytesensor elements, architectures and methods for making and using theseelements that are disclosed herein can be used to establish a variety oflayered sensor structures. Such sensors of the invention exhibit asurprising degree of flexibility and versatility, characteristics whichallow a wide variety of sensor configurations to be designed to examinea wide variety of analyte species.

Specific aspects of embodiments of the invention are discussed in detailin the following sections.

Typical Elements, Configurations and Analyte Sensors of the Invention

Optimized Sensor Elements of the Invention

A wide variety of sensors and sensor elements are known in the artincluding amperometric sensors used to detect and/or measure biologicalanalytes such as glucose. Many glucose sensors are based on an oxygen(Clark-type) amperometric transducer (see, e.g. Yang et al.,Electroanalysis 1997, 9, No. 16: 1252-1256; Clark et al., Ann. N.Y.Acad. Sci. 1962, 102, 29; Updike et al., Nature 1967, 214,986; andWilkins et al., Med. Engin. Physics, 1996, 18, 273.3-51). A number of invivo glucose sensors utilize hydrogen peroxide-based amperometrictransducers because such transducers are relatively easy to fabricateand can readily be miniaturized using conventional technology. Oneproblem associated with the use of certain amperometric transducers,however, include a suboptimal reaction stoichiometry. As discussed indetail below, these problems are addressed by using the polycarbonatepolymeric membrane(s) disclosed herein, membranes which can modulate thetransport properties of different compounds whose reaction creates asignal at the hydrogen peroxide-based amperometric transducing element.Consequently, these membranes can be used for example with a variety ofH₂O₂ based analyte sensors that benefit from optimized reactionstoichiometries.

As noted above, embodiments of the invention include sensor membranesmade from reaction mixtures form to include limiting amounts of catalystand/or polycarbonate polymer compositions. As is known in the art, apolymer comprises a long or larger molecule consisting of a chain ornetwork of many repeating units, formed by chemically bonding togethermany identical or similar small molecules called monomers. A copolymeror heteropolymer is a polymer derived from two (or more) monomericspecies, as opposed to a homopolymer where only one monomer is used.Copolymers may also be described in terms of the existence of orarrangement of branches in the polymer structure. Linear copolymersconsist of a single main chain whereas branched copolymers consist of asingle main chain with one or more polymeric side chains. Sensormembranes made from polycarbonate polymeric compositions disclosedherein can optimize analyte sensor function including sensorsensitivity, stability and hydration profiles. In addition, byoptimizing the stoichiometry of reactant species over a range of sensortemperatures, the membranes disclosed herein can optimize the chemicalreactions that produce the critical measurable signals that correlatewith the levels of an analyte of interest (e.g. glucose). The followingsections describe illustrative sensor elements, sensor configurationsand methodological embodiments of the invention.

The polymeric materials disclosed herein are useful as biocompatiblemembranes in a variety of contexts, for example as a glucose limitingmembranes (GLM). However, polymers typically degrade over time due tooxidation, UV light, heat, hydrolysis, or other processes. In thiscontext, we have discovered that trace amount of tin catalyst residuefrom the polymeric mixture used to form biocompatible membranes such asthe GLM can speed up GLM degradation overtime. For this reason, oldersensors may perform slightly worse than fresh-made sensors due to thegradual degradation of the GLM. While not being bound by a specificscientific theory or mechanism of action, it is believed that traceamount of tin catalyst residue in GLM may further trigger immuneresponse and sensitivity loss. In this context, we have furtherdiscovered that reducing the amount of tin catalyst (e.g. by 50%) usedin GLM synthesis process unexpectedly generates membranes having anincreased resistance to thermal degradation, and improves the quality(biocompatibility and thermal stability) of the GLM as well as glucosesensor in vivo performance. This greater GLM stability further resultsin longer sensor shelf life and a more biocompatible sensor, without anysignificant manufacturing process change.

The invention disclosed herein has a number of embodiments. Oneembodiment of the invention is a method of increasing the thermalstability of a biocompatible membrane formed by a reaction mixturecomprising a diisocyanate, a hydrophilic polymer comprising ahydrophilic diol or hydrophilic diamine, a siloxane having an amino,hydroxyl or carboxylic acid functional group at a terminus; and acatalyst. In this methodology, the reaction mixture is formed so thatthe catalyst is present in the reaction mixture in amounts less than0.2% of reaction mixture components (e.g. 0.1%), thereby increasing thethermal stability of the biocompatible membrane as compared to acomparable membrane formed from a reaction mixture where the catalyst ispresent in the formulation in amounts greater than or equal to 0.2% ofthe reaction mixture. Optionally the reaction mixture uses an organicsolvent such as tetrahydrofuran (e.g. at 60° C.) and further comprisesadditional components such as a polycarbonate diol. The thermalstability of various biocompatible membrane made in this way can bemeasured by a variety of art accepted practices, for example byobserving changes in molecular weight of the biocompatible membranemaintained at a temperature of 60° C. over at least 3, 5 or 7 days (seee.g. FIG. 7 ).

In certain embodiments of the invention, a tin catalyst (e.g. Dibutyltinbis(2-ethylhexanoate)) is present in the reaction mixture in amountsless than 0.19%, 0.17%, 0.15%, 0.13%, or 0.11% of the reaction mixture(e.g. 0.1%). In some embodiments of the invention, the diisocyanatecomprises a hexamethylene diisocyanate and/or a methylene diphenyldiisocyanate, and/or the hydrophilic polymer comprising a hydrophilicdiol or hydrophilic diamine comprises a JEFFAMINE, and/or the siloxanehaving an amino, hydroxyl or carboxylic acid functional group at aterminus comprises a polydimethylsiloxane, and/or the polycarbonate diolcomprises a (poly(1,6-hexyle carbonate) diol and/or a or poly(1,6hexyl-1,5 pentyl carbonate) diol. For example, in certain embodiments ofthe invention, the diisocyanate comprise from 17% to 23% weight percenthexamethylene diisocyanate and from 0% to 8.5% weight percent methylenediphenyl diisocyanate, and the JEFFAMINE comprises from 28% to 51%weight percent JEFFAMINE 600 and/or JEFFAMINE 900, and thepolydimethylsiloxane comprises from 14% to 48% weight percentpolydimethylsiloxane-A15), and the polycarbonate diol comprises from7.5% to 19% weight percent (poly(1,6-hexyle carbonate) diol. In aspecific illustrative embodiment, the diisocyanate comprises about 22%hexamethylene diisocyanate and about 3.5% methylene diphenyldiisocyanate, the JEFFAMINE comprises about 45% JEFFAMINE 600 and/orJEFFAMINE 900, the polydimethylsiloxane comprises about 22.5%polydimethylsiloxane-A15), and the polycarbonate diol comprises about7.5% (poly(1,6-hexyle carbonate) diol. Optionally in this method, wateris added as a chain extender in the reaction mixture of thepolyurea-urethane copolymer. Illustrative reaction mixtures of theinvention are shown in Tables 1 and 2 below.

TABLE 1 Tin catalyst Dibutyltin Jeffa- UH- bis(2- mine 100 ethylhex- 600PDMS diol MDI HMDI anoate) Poly- 44.90% 22.50% 7.50% 3.40% 21.70% 0.20%carbonate GLM (control) Poly- 44.90% 22.50% 7.50% 3.40% 21.70% 0.10%carbonate GLM with 50% Tin catalyst

TABLE 2 Tin catalyst Jeffamine Dibutyltin 600 PDMS HMDIbis(2-ethylhexanoate) 2XGLM 40.80% 36.70% 22.50% 0.20% (control) 2XGLMwith 40.80% 36.70% 22.50% 0.10% 50% Tin

Another embodiment of the invention is an amperometric analyte sensorcomprising a base layer, a conductive layer disposed on the base layerand comprising a working electrode, an analyte sensing layer disposed onthe conductive layer, and an analyte modulating layer disposed on theanalyte sensing layer. In this embodiment, the analyte modulating layeris formed by a reaction mixture comprising a diisocyanate, a hydrophilicpolymer comprising a hydrophilic diol or hydrophilic diamine, a siloxanehaving an amino, hydroxyl or carboxylic acid functional group at aterminus, and a catalyst. In this embodiment, the amount of catalystpresent in the reaction mixture in amounts less than 0.2% of reactionmixture components so that the analyte modulating layer exhibits agreater thermal stability than a comparable analyte modulating layerformed from a reaction mixture where the catalyst is present in theformulation in amounts greater than or equal to 0.2% of the reactionmixture. Optionally the reaction mixture further comprises additionalcomponents such as a polycarbonate diol.

In typical embodiments, the analyte sensor is a glucose sensor that isimplantable in vivo. Optionally, the analyte sensor further comprises atleast one of: a protein layer disposed on the analyte sensing layer, ora cover layer disposed on the analyte sensor apparatus, and the coverlayer comprises an aperture positioned on the cover layer so as tofacilitate an analyte present in an in vivo environment from contactingand diffusing through an analyte modulating layer; and contacting theanalyte sensing layer. In certain of these analyte sensors, theconductive layer comprises a plurality of electrodes including a workingelectrode, a counter electrode and a reference electrode, for example anembodiment where the conductive layer comprises a plurality of workingelectrodes and/or counter electrodes and/or reference electrodes; andoptionally the plurality of working, counter and reference electrodesare grouped together as a unit and positionally distributed on theconductive layer in a repeating pattern of units.

Yet another embodiment of the invention is a method of making an analytesensor for implantation within a mammal. This methodological embodimentcomprises the steps of providing a base layer, forming a conductivelayer on the base layer, wherein the conductive layer includes a workingelectrode, forming an analyte sensing layer on the conductive layer,wherein the analyte sensing layer includes an oxidoreductase, and thenforming an analyte modulating layer on the analyte sensing layer. Inthis embodiment, the analyte modulating layer is formed by a reactionmixture comprising a diisocyanate, a hydrophilic polymer comprising ahydrophilic diol or hydrophilic diamine, a siloxane having an amino,hydroxyl or carboxylic acid functional group at a terminus; and acatalyst present in the reaction mixture in amounts less than 0.2% (e.g.0.1%) of reaction mixture components so that the reaction mixtureexhibits a greater thermal stability than a comparable analytemodulating layer formed from a reaction mixture where the catalyst ispresent in the formulation in amounts greater than or equal to 0.2% ofthe reaction mixture. Optionally the reaction mixture further comprisesadditional components such as a polycarbonate diol.

In certain method of making an analyte sensor for implantation within amammal, the diisocyanate comprises a hexamethylene diisocyanate and/or amethylene diphenyl diisocyanate, the JEFFAMINE comprises about 45%JEFFAMINE 600 and/or JEFFAMINE 900, the polydimethylsiloxane comprisesabout 22.5% polydimethylsiloxane-A15), and the polycarbonate diolcomprises about 7.5% (poly(1,6-hexyle carbonate) diol. Typically in thisembodiment, the catalyst (e.g. Dibutyltin bis(2-ethylhexanoate)) ispresent in the reaction mixture in amounts less than 0.19%, 0.17%,0.15%, 0.13%, or 0.11% of the reaction mixture (e.g. about 0.1%).

Certain amperometric sensor design used with embodiments of theinvention comprise a plurality of layered elements including for examplea base layer having an electrode, an analyte sensing layer (e.g. onecomprising glucose oxidase) and an analyte modulating layer thatfunctions in analyte diffusion control (e.g. to modulate the amounts ofglucose and oxygen exposed to the analyte sensing layer). One suchsensor embodiment is shown in FIG. 1 . Layered sensor designs thatincorporate the polycarbonate polymeric compositions disclosed herein asthe analyte modulating layer exhibit a constellation of materialproperties that overcome challenges observed in a variety of sensorsincluding electrochemical glucose sensors that are implanted in vivo.For example, sensors designed to measure analytes in aqueousenvironments (e.g. those implanted in vivo) typically require wetting ofthe layers prior to and during the measurement of accurate analytereading. Because the properties of a material can influence the rate atwhich it hydrates, the material properties of membranes used in aqueousenvironments ideally will facilitate sensor wetting to, for example,minimize the time period between the sensor's introduction into anaqueous environment and its ability to provide accurate signals thatcorrespond to the concentrations of an analyte in that environment.Embodiments of the invention that comprise polycarbonate polymericcompositions address such issues by facilitating sensor hydration.

Moreover, with electrochemical glucose sensors that utilize the chemicalreaction between glucose and glucose oxidase to generate a measurablesignal, the material of the analyte modulating layer should notexacerbate (and ideally should diminish) what is known in the art as the“oxygen deficit problem”. Specifically, because glucose oxidase basedsensors require both oxygen (02) as well as glucose to generate asignal, the presence of an excess of oxygen relative to glucose, isnecessary for the operation of a glucose oxidase based glucose sensor.However, because the concentration of oxygen in subcutaneous tissue ismuch less than that of glucose, oxygen can be the limiting reactant inthe reaction between glucose, oxygen, and glucose oxidase in a sensor, asituation which compromises the sensor's ability to produce a signalthat is strictly dependent on the concentration of glucose. In thiscontext, because the properties of a material can influence the rate atwhich compounds diffuse through that material to the site of ameasurable chemical reaction, the material properties of an analytemodulating layer used in electrochemical glucose sensors that utilizethe chemical reaction between glucose and glucose oxidase to generate ameasurable signal, should not for example, favor the diffusion ofglucose over oxygen in a manner that contributes to the oxygen deficitproblem. Embodiments of the invention that comprise the polycarbonatepolymeric compositions disclosed herein do not contribute to, andinstead function to ameliorate, the oxygen deficit problem.

In addition, sensor designs that use the polycarbonate polymericcompositions disclosed herein as a analyte modulating layer can alsoovercome complications observed with the use of sensor materials thatcan exhibit different diffusion profiles (e.g. a rate at which ananalyte diffuses therethrough) at different temperatures. In particular,for optimized sensor performance, sensor signal output over a range oftemperatures should be determined only by the levels of analyte ofinterest (e.g. glucose), and not by any co-substrates (e.g. O₂) orkinetically controlled parameters (e.g. diffusion). As is known in theart however, the diffusion of compounds through a polymeric matrix canbe temperature dependent. In situations where an analyte (e.g. glucose)diffuses through a polymer to react a site where it reacts with anothercompound (e.g. glucose oxidase), such temperature dependent diffusionprofiles can influence the stoichiometry of the reaction relied upon togenerate the sensor signal, thereby confounding artisans' efforts tomake sensor signal output depend only on the concentration of an analyteof interest over a range of temperatures. Analyte modulatingcompositions made from materials having an analyte (e.g. glucose)diffusion profile that is stable over a range of temperatures (e.g. from22 to 40 degrees centigrade) consequently address such issues.

The invention disclosed herein provides polycarbonate polymericcompositions useful for example as membranes for biosensors such asamperometric glucose sensors. Embodiments of the invention include forexample a sensor having a plurality of layered elements including ananalyte limiting membrane comprising a polycarbonate polymericcomposition. Such polymeric membranes are particularly useful in theconstruction of electrochemical sensors for in vivo use. The membraneembodiments of the invention allow for a combination of desirableproperties including: an enhanced hydration profile as well as apermeability to molecules such as glucose that is stable over a range oftemperatures. In addition, these polymeric membranes exhibit goodmechanical properties for use as an outer polymeric membrane.Consequently, glucose sensors that incorporate such polymeric membranesshow a highly desirable in-vivo performance profile.

Embodiments of the invention include both materials (e.g. polycarbonatepolymeric compositions) as well as architectures that designed tofacilitate sensor performance. For example, in certain embodiments ofthe invention, the conductive layer comprises a plurality of workingelectrodes and/or counter electrodes and/or reference electrodes (e.g. 3working electrodes, a reference electrode and a counter electrode), inorder to, for example, avoid problems associated with poor sensorhydration and/or provide redundant sensing capabilities. Optionally, theplurality of working, counter and reference electrodes are configuredtogether as a unit and positionally distributed on the conductive layerin a repeating pattern of units. In certain embodiments of theinvention, the base layer is made from a flexible material that allowsthe sensor to twist and bend when implanted in vivo; and the electrodesare grouped in a configuration that facilitates an in vivo fluidcontacting at least one of working electrode as the sensor apparatustwists and bends when implanted in vivo. In some embodiments, theelectrodes are grouped in a configuration that allows the sensor tocontinue to function if a portion of the sensor having one or moreelectrodes is dislodged from an in vivo environment and exposed to an exvivo environment. Typically, the sensor is operatively coupled to asensor input capable of receiving a signal from the sensor that is basedon a sensed analyte; and a processor coupled to the sensor input,wherein the processor is capable of characterizing one or more signalsreceived from the sensor. In some embodiments of the invention, a pulsedvoltage is used to obtain a signal from one or more electrodes of asensor.

The sensors disclosed herein can be made from a wide variety ofmaterials known in the art. In one illustrative embodiment of theinvention, the analyte modulating layer comprises apolyurethane/polyurea polymer formed from a mixture comprising: adiisocyanate; a hydrophilic polymer comprising a hydrophilic diol orhydrophilic diamine; and a siloxane having an amino, hydroxyl orcarboxylic acid functional group at a terminus; with this polymer thenpolycarbonate with a branched acrylate polymer formed from a mixturecomprising: a butyl, propyl, ethyl or methyl-acrylate; anamino-acrylate; a siloxane-acrylate; and a poly(ethyleneoxide)-acrylate. Optionally, additional materials can be included inthese polymeric blends. For example, certain embodiments of the branchedacrylate polymer are formed from a reaction mixture that includes ahydroxyl-acrylate compound (e.g. 2-hydroxyethyl methacrylate).

As used herein, the term “polyurethane/polyurea polymer” refers to apolymer containing urethane linkages, urea linkages or combinationsthereof. As is known in the art, polyurethane is a polymer consisting ofa chain of organic units joined by urethane (carbamate) links.Polyurethane polymers are typically formed through step-growthpolymerization by reacting a monomer containing at least two isocyanatefunctional groups with another monomer containing at least two hydroxyl(alcohol) groups in the presence of a catalyst. Polyurea polymers arederived from the reaction product of an isocyanate component and adiamine. Typically, such polymers are formed by combining diisocyanateswith alcohols and/or amines. For example, combining isophoronediisocyanate with PEG 600 and aminopropyl polysiloxane underpolymerizing conditions provides a polyurethane/polyurea compositionhaving both urethane (carbamate) linkages and urea linkages. Suchpolymers are well known in the art and described for example in U.S.Pat. Nos. 5,777,060, 5,882,494 and 6,632,015, and PCT publications WO96/30431; WO 96/18115; WO 98/13685; and WO 98/17995, the contents ofeach of which is incorporated by reference.

The polyurethane/polyurea compositions of the invention are preparedfrom biologically acceptable polymers whose hydrophobic/hydrophilicbalance can be varied over a wide range to control the ratio of thediffusion coefficient of oxygen to that of glucose, and to match thisratio to the design requirements of electrochemical glucose sensorsintended for in vivo use. Such compositions can be prepared byconventional methods by the polymerization of monomers and polymersnoted above. The resulting polymers are soluble in solvents such asacetone or ethanol and may be formed as a membrane from solution by dip,spray or spin coating.

Diisocyanates useful in this embodiment of the invention are those whichare typically those which are used in the preparation of biocompatiblepolyurethanes. Such diisocyanates are described in detail in Szycher,SEMINAR ON ADVANCES IN MEDICAL GRADE POLYURETHANES, TechnomicPublishing, (1995) and include both aromatic and aliphaticdiisocyanates. Examples of suitable aromatic diisocyanates includetoluene diisocyanate, 4,4′-diphenylmethane diisocyanate,3,3′-dimethyl-4,4′-biphenyl diisocyanate, naphthalene diisocyanate andparaphenylene diisocyanate. Suitable aliphatic diisocyanates include,for example, 1,6hexamethylene diisocyanate (HDI), trimethylhexamethylenediisocyanate (TMDI), trans1,4-cyclohexane diisocyanate (CHDI),1,4-cyclohexane bis(methylene isocyanate) (BDI), 1,3-cyclohexanebis(methylene isocyanate) (H₆ XDI), isophorone diisocyanate (IPDI) and4,4′-methylenebis(cyclohexyl isocyanate) (H₂ MDI). In some embodiments,the diisocyanate is isophorone diisocyanate, 1,6-hexamethylenediisocyanate, or 4,4′methylenebis(cyclohexyl isocyanate). A number ofthese diisocyanates are available from commercial sources such asAldrich Chemical Company (Milwaukee, Wis., USA) or can be readilyprepared by standard synthetic methods using literature procedures.

The quantity of diisocyanate used in the reaction mixture for thepolyurethane/polyurea polymer compositions is typically about 50 mol %relative to the combination of the remaining reactants. Moreparticularly, the quantity of diisocyanate employed in the preparationof the polyurethane/polyurea polymer will be sufficient to provide atleast about 100% of the —NCO groups necessary to react with the hydroxylor amino groups of the remaining reactants. For example, a polymer whichis prepared using x moles of diisocyanate, will use a moles of ahydrophilic polymer (diol, diamine or combination), b moles of asilicone polymer having functionalized termini, and c moles of a chainextender, such that x=a+b+c, with the understanding that c can be zero.

Another reactant used in the preparation of the polyurethane/polyureapolymers described herein is a hydrophilic polymer. The hydrophilicpolymer can be a hydrophilic diol, a hydrophilic diamine or acombination thereof. The hydrophilic diol can be a poly(alkylene)glycol,a polyester-based polyol, or a polycarbonate polyol. As used herein, theterm “poly(alkylene)glycol” refers to polymers of lower alkylene glycolssuch as poly(ethylene)glycol, poly(propylene)glycol andpolytetramethylene ether glycol (PTMEG). The term “polyester-basedpolyol” refers to a polymer in which the R group is a lower alkylenegroup such as ethylene, 1,3-propylene, 1,2-propylene,1,4-butylene,2,2-dimethyl-1,3-propylene, and the like (e.g. as depictedin FIG. 4 of U.S. Pat. No. 5,777,060). One of skill in the art will alsounderstand that the diester portion of the polymer can also vary fromthe six-carbon diacid shown. For example, while FIG. 4 of U.S. Pat. No.5,777,060 illustrates an adipic acid component, the present inventionalso contemplates the use of succinic acid esters, glutaric acid estersand the like. The term “polycarbonate polyol” refers those polymershaving hydroxyl functionality at the chain termini and ether andcarbonate functionality within the polymer chain. The alkyl portion ofthe polymer will typically be composed of C2 to C4 aliphatic radicals,or in some embodiments, longer chain aliphatic radicals, cycloaliphaticradicals or aromatic radicals. The term “hydrophilic diamines” refers toany of the above hydrophilic diols in which the terminal hydroxyl groupshave been replaced by reactive amine groups or in which the terminalhydroxyl groups have been derivatized to produce an extended chainhaving terminal amine groups. For example, a some hydrophilic diamine isa “diamino poly(oxyalkylene)” which is poly(alkylene)glycol in which theterminal hydroxyl groups are replaced with amino groups. The term“diamino poly(oxyalkylene” also refers to poly(alkylene)glycols whichhave aminoalkyl ether groups at the chain termini. One example of asuitable diamino poly(oxyalkylene) is poly(propyleneglycol)bis(2-aminopropyl ether). A number of the above polymers can beobtained from Aldrich Chemical Company. Alternatively, conventionalmethods known in the art can be employed for their synthesis.

The amount of hydrophilic polymer which is used to make the linearpolymer compositions will typically be about 10% to about 80% by molerelative to the diisocyanate which is used. Typically, the amount isfrom about 20% to about 60% by mole relative to the diisocyanate. Whenlower amounts of hydrophilic polymer are used, it is common to include achain extender.

Silicone containing polyurethane/polyurea polymers which are useful inthe present invention are typically linear, have excellent oxygenpermeability and essentially no glucose permeability. Typically, thesilicone polymer is a polydimethylsiloxane having two reactivefunctional groups (i.e., a functionality of 2). The functional groupscan be, for example, hydroxyl groups, amino groups or carboxylic acidgroups, but are typically hydroxyl or amino groups. In some embodiments,combinations of silicone polymers can be used in which a first portioncomprises hydroxyl groups and a second portion comprises amino groups.Typically, the functional groups are positioned at the chain termini ofthe silicone polymer. A number of suitable silicone polymers arecommercially available from such sources as Dow Chemical Company(Midland, Mich., USA) and General Electric Company (Silicones Division,Schenectady, N.Y., USA). Still others can be prepared by generalsynthetic methods known in the art (see, e.g. U.S. Pat. No. 5,777,060),beginning with commercially available siloxanes (United ChemicalTechnologies, Bristol. Pa., USA). For use in the present invention, thesilicone polymers will typically be those having a molecular weight offrom about 400 to about 10,000, more typically those having a molecularweight of from about 2000 to about 4000. The amount of silicone polymerwhich is incorporated into the reaction mixture will depend on thedesired characteristics of the resulting polymer from which thebiocompatible membrane is formed. For those compositions in which alower glucose penetration is desired, a larger amount of siliconepolymer can be employed. Alternatively, for compositions in which ahigher glucose penetration is desired, smaller amounts of siliconepolymer can be employed. Typically, for a glucose sensor, the amount ofsiloxane polymer will be from 10% to 90% by mole relative to thediisocyanate. Typically, the amount is from about 20% to 60% by molerelative to the diisocyanate.

In one group of embodiments, the reaction mixture for the preparation ofbiocompatible membranes will also contain a chain extender which is analiphatic or aromatic diol, an aliphatic or aromatic diamine,alkanolamine, or combinations thereof (e.g. as depicted in FIG. 8 ofU.S. Pat. No. 5,777,060)). Examples of suitable aliphatic chainextenders include ethylene glycol, propylene glycol, 1,4-butanediol,1,6-hexanediol, ethanolamine, ethylene diamine, butane diamine,1,4-cyclohexanedimethanol. Aromatic chain extenders include, forexample, para-di(2-hydroxyethoxy)benzene,meta-di(2-hydroxyethoxy)benzene, Ethacure 100® (a mixture of two isomersof 2,4-diamino-3,5-diethyltoluene), Ethacure 300®(2,4-diamino-3,5-di(methylthio)toluene),3,3′-dichloro-4,4′diaminodiphenylmethane, Polacure® 740M (trimethyleneglycol bis(para-aminobenzoate)ester), and methylenedianiline.Incorporation of one or more of the above chain extenders typicallyprovides the resulting biocompatible membrane with additional physicalstrength, but does not substantially increase the glucose permeabilityof the polymer. Typically, a chain extender is used when lower (i.e.,10-40 mol %) amounts of hydrophilic polymers are used. In particularlysome compositions, the chain extender is diethylene glycol which ispresent in from about 40% to 60% by mole relative to the diisocyanate.

Polymerization of the above reactants can be carried out in bulk or in asolvent system. Use of a catalyst is some, though not required. Suitablecatalysts include dibutyltin bis(2-ethylhexanoate) (CAS #: 2781-10-4),dibutyltin diacetate, triethylamine and combinations thereof. Typicallydibutyltin bis(2-ethylhexanoate is used as the catalyst. The typicalamount of this catalyst used is in the formulation is from 0.05% to 0.2%(w/w ratio). Bulk polymerization is typically carried out at an initialtemperature of about 25° C. (ambient temperature) to about 50° C., inorder to insure adequate mixing of the reactants. Upon mixing of thereactants, an exotherm is typically observed, with the temperaturerising to about 90-120° C. After the initial exotherm, the reactionflask can be heated at from 75° C. to 125° C., with 90°. C. to 100° C.being an exemplary temperature range. Heating is usually carried out forone to two hours. Solution polymerization can be carried out in asimilar manner. Solvents which are suitable for solution polymerizationinclude dimethylformamide, dimethyl sulfoxide, dimethylacetamide,halogenated solvents such as 1,2,3-trichloropropane, and ketones such as4-methyl-2-pentanone. Typically, THF is used as the solvent. Whenpolymerization is carried out in a solvent, heating of the reactionmixture is typically carried out for three to four hours.

Polymers prepared by bulk polymerization are typically dissolved indimethylformamide and precipitated from water. Polymers prepared insolvents that are not miscible with water can be isolated by vacuumstripping of the solvent. These polymers are then dissolved indimethylformamide and precipitated from water. After thoroughly washingwith water, the polymers can be dried in vacuo at about 50° C. toconstant weight.

Preparation of the membranes can be completed by dissolving the driedpolymer in a suitable solvent and cast a film onto a glass plate. Theselection of a suitable solvent for casting will typically depend on theparticular polymer as well as the volatility of the solvent. Typically,the solvent is THF, CHCl₃, CH₂Cl₂, DMF, IPA or combinations thereof.More typically, the solvent is THF or DMF/CH₂Cl₂ ( 2/98 volume %). Thesolvent is removed from the films, the resulting membranes are hydratedfully, their thicknesses measured and water pickup is determined.Membranes which are useful in the present invention will typically havea water pickup of about 20 to about 100%, typically 30 to about 90%, andmore typically 40 to about 80%, by weight.

Oxygen and glucose diffusion coefficients can also be determined for theindividual polymer compositions as well as the polycarbonate polymericmembranes of the present invention. Methods for determining diffusioncoefficients are known to those of skill in the art, and examples areprovided below. Certain embodiments of the biocompatible membranesdescribed herein will typically have an oxygen diffusion coefficient(D_(oxygen)) of about 0.1×10⁻⁶ cm²/sec to about 2.0×10⁻⁶ cm²/sec and aglucose diffusion coefficient (D_(glucose)) of about 1×10⁻⁹ cm²/sec toabout 500×10⁻⁹ cm²/sec. More typically, the glucose diffusioncoefficient is about 10×10⁻⁹ cm²/sec to about 200×10⁻⁹ cm²/sec.

Typical Combinations of Sensor Elements

Embodiments of the invention further include sensors comprising thepolycarbonate polymeric compositions disclosed herein in combinationwith other sensor elements such as an interference rejection membrane(e.g. an interference rejection membrane as disclosed in U.S. patentapplication Ser. No. 12/572,087, the contents of which are incorporatedby reference). One such embodiment of the invention is an interferencerejection membrane comprising methacrylate polymers having a molecularweight between 100 and 1000 kilodaltons, wherein the methacrylatepolymers are crosslinked by a hydrophilic crosslinking agent such as anorganofunctional dipodal alkoxysilane. Another embodiment of theinvention is an interference rejection membrane comprising primary aminepolymers having a molecular weight between 4,000 Daltons and 500kilodaltons, wherein the primary amine polymers are crosslinked by ahydrophilic crosslinking agent such as glutaraldehyde. Typically theseinterference rejection membranes coat a hydrogen peroxide transducingcomposition. In an illustrative embodiment, the hydrogen peroxidetransducing composition comprises an electrode; and the crosslinkedinterference rejection membrane is coated on the electrode in a layerbetween 0.1 μm and 1.0 μm thick.

In some embodiments of the invention, an element of the sensor apparatussuch as an electrode or an aperture is designed to have a specificconfiguration and/or is made from a specific material and/or ispositioned relative to the other elements so as to facilitate a functionof the sensor. In one such embodiment of the invention, a workingelectrode, a counter electrode and a reference electrode arepositionally distributed on the base and/or the conductive layer in aconfiguration that facilitates sensor start up and/or maintains thehydration of the working electrode, the counter electrode and/or thereference electrode when the sensor apparatus is placed in contact witha fluid comprising the analyte (e.g. by inhibiting shadowing of anelectrode, a phenomena which can inhibit hydration and capacitivestart-up of a sensor circuit). Typically such embodiments of theinvention facilitate sensor start-up and/or initialization.

Optionally embodiments of the apparatus comprise a plurality of workingelectrodes and/or counter electrodes and/or reference electrodes (e.g. 3working electrodes, a reference electrode and a counter electrode), inorder to, for example, provide redundant sensing capabilities. Certainembodiments of the invention comprising a single sensor. Otherembodiments of the invention comprise multiple sensors. In someembodiments of the invention, a pulsed voltage is used to obtain asignal from one or more electrodes of a sensor. Optionally, theplurality of working, counter and reference electrodes are configuredtogether as a unit and positionally distributed on the conductive layerin a repeating pattern of units. In certain embodiments of theinvention, the elongated base layer is made from a flexible materialthat allows the sensor to twist and bend when implanted in vivo; and theelectrodes are grouped in a configuration that facilitates an in vivofluid contacting at least one of working electrode as the sensorapparatus twists and bends when implanted in vivo. In some embodiments,the electrodes are grouped in a configuration that allows the sensor tocontinue to function if a portion of the sensor having one or moreelectrodes is dislodged from an in vivo environment and exposed to an exvivo environment.

In certain embodiments of the invention comprising multiple sensors,elements such as the sensor electrodes are organized/disposed within aflex-circuit assembly. In such embodiments of the invention, thearchitecture of the sensor system can be designed so that a first sensordoes not influence a signal etc. generated by a second sensor (and viceversa); and so that the first and second sensors sense from separatetissue envelopes; so the signals from separate sensors do not interact.At the same time, in typical embodiments of the invention the sensorswill be spaced at a distance from each other so that allows them to beeasily packaged together and/or adapted to be implanted via a singleinsertion action. One such embodiment of the invention is an apparatusfor monitoring an analyte in a patient, the apparatus comprising: a baseelement adapted to secure the apparatus to the patient; a first piercingmember coupled to and extending from the base element; a firstelectrochemical sensor operatively coupled to the first piercing memberand comprising a first electrochemical sensor electrode for determiningat least one physiological characteristic of the patient at a firstelectrochemical sensor placement site; a second piercing member coupledto and extending from the base element; a second electrochemical sensoroperatively coupled to the second piercing member and comprising asecond electrochemical sensor electrode for determining at least onephysiological characteristic of the patient at a second electrochemicalsensor placement site. In such embodiments of the invention, at leastone physiological characteristic monitored by the first or the secondelectrochemical sensor comprises a concentration of a naturallyoccurring analyte in the patient; the first piercing member disposes thefirst electrochemical sensor in a first tissue compartment of thepatient and the second piercing member disposes the secondelectrochemical sensor in a second tissue compartment of the patient;and the first and second piercing members are disposed on the base in aconfiguration selected to avoid a physiological response that can resultfrom implantation of the first electrochemical sensor from altering asensor signal generated by the second electrochemical sensor.

Various elements of the sensor apparatus can be disposed at a certainlocation in the apparatus and/or configured in a certain shape and/or beconstructed from a specific material so as to facilitate strength and/orfunction of the sensor. One embodiment of the invention includes anelongated base comprised of a polyimmide or dielectric ceramic materialthat facilitates the strength and durability of the sensor. In certainembodiments of the invention, the structural features and/or relativeposition of the working and/or counter and/or reference electrodes isdesigned to influence sensor manufacture, use and/or function.Optionally, the sensor is operatively coupled to a constellation ofelements that comprise a flex-circuit (e.g. electrodes, electricalconduits, contact pads and the like). One embodiment of the inventionincludes electrodes having one or more rounded edges so as to inhibitdelamination of a layer disposed on the electrode (e.g. an analytesensing layer comprising glucose oxidase).

In certain embodiments of the invention, an electrode of the apparatuscomprises a platinum composition and the apparatus further comprises atitanium composition disposed between the elongated base layer and theconductive layer. Optionally in such embodiments, apparatus furthercomprises a gold composition disposed between the titanium compositionand the conductive layer. Such embodiments of the invention typicallyexhibit enhanced bonding between layered materials within the sensorand/or less corrosion and/or improved biocompatibility profiles. Relatedembodiments of the invention include methods for inhibiting corrosion ofa sensor element and/or method for improving the biocompatibility of asensor embodiments of the invention (e.g. one constructed to use suchmaterials).

In typical embodiments of the invention, the sensor is operativelycoupled to further elements (e.g. electronic components) such aselements designed to transmit and/or receive a signal, monitors,processors and the like as well as devices that can use sensor data tomodulate a patient's physiology such as medication infusion pumps. Forexample, in some embodiments of the invention, the sensor is operativelycoupled to a sensor input capable of receiving a signal from the sensorthat is based on a sensed physiological characteristic value in themammal; and a processor coupled to the sensor input, wherein theprocessor is capable of characterizing one or more signals received fromthe sensor. A wide variety of sensor configurations as disclosed hereincan be used in such systems. Optionally, for example, the sensorcomprises three working electrodes, one counter electrode and onereference electrode. In certain embodiments, at least one workingelectrode is coated with an analyte sensing layer comprising glucoseoxidase and at least one working electrode is not coated with an analytesensing layer comprising glucose oxidase.

Diagrammatic Illustration of Typical Sensor Configurations

FIG. 1 illustrates a cross-section of a typical sensor embodiment 100 ofthe present invention. This sensor embodiment is formed from a pluralityof components that are typically in the form of layers of variousconductive and non-conductive constituents disposed on each otheraccording to art accepted methods and/or the specific methods of theinvention disclosed herein. The components of the sensor are typicallycharacterized herein as layers because, for example, it allows for afacile characterization of the sensor structure shown in FIG. 1 .Artisans will understand however, that in certain embodiments of theinvention, the sensor constituents are combined such that multipleconstituents form one or more heterogeneous layers. In this context,those of skill in the art understand that the ordering of the layeredconstituents can be altered in various embodiments of the invention.

The embodiment shown in FIG. 1 includes a base layer 102 to support thesensor 100. The base layer 102 can be made of a material such as a metaland/or a ceramic and/or a polymeric substrate, which may beself-supporting or further supported by another material as is known inthe art. Embodiments of the invention include a conductive layer 104which is disposed on and/or combined with the base layer 102. Typicallythe conductive layer 104 comprises one or more electrodes. An operatingsensor 100 typically includes a plurality of electrodes such as aworking electrode, a counter electrode and a reference electrode. Otherembodiments may also include a plurality of working and/or counterand/or reference electrodes and/or one or more electrodes that performsmultiple functions, for example one that functions as both as areference and a counter electrode.

As discussed in detail below, the base layer 102 and/or conductive layer104 can be generated using many known techniques and materials. Incertain embodiments of the invention, the electrical circuit of thesensor is defined by etching the disposed conductive layer 104 into adesired pattern of conductive paths. A typical electrical circuit forthe sensor 100 comprises two or more adjacent conductive paths withregions at a proximal end to form contact pads and regions at a distalend to form sensor electrodes. An electrically insulating cover layer106 such as a polymer coating can be disposed on portions of the sensor100. Acceptable polymer coatings for use as the insulating protectivecover layer 106 can include, but are not limited to, non-toxicbiocompatible polymers such as silicone compounds, polyimides,biocompatible solder masks, epoxy acrylate copolymers, or the like. Inthe sensors of the present invention, one or more exposed regions orapertures 108 can be made through the cover layer 106 to open theconductive layer 104 to the external environment and to, for example,allow an analyte such as glucose to permeate the layers of the sensorand be sensed by the sensing elements. Apertures 108 can be formed by anumber of techniques, including laser ablation, tape masking, chemicalmilling or etching or photolithographic development or the like. Incertain embodiments of the invention, during manufacture, a secondaryphotoresist can also be applied to the protective layer 106 to definethe regions of the protective layer to be removed to form theaperture(s) 108. The exposed electrodes and/or contact pads can alsoundergo secondary processing (e.g. through the apertures 108), such asadditional plating processing, to prepare the surfaces and/or strengthenthe conductive regions.

In the sensor configuration shown in FIG. 1 , an analyte sensing layer110 (which is typically a sensor chemistry layer, meaning that materialsin this layer undergo a chemical reaction to produce a signal that canbe sensed by the conductive layer) is disposed on one or more of theexposed electrodes of the conductive layer 104. In the sensorconfiguration shown in FIG. 2B, an interference rejection membrane 120is disposed on one or more of the exposed electrodes of the conductivelayer 104, with the analyte sensing layer 110 then being disposed onthis interference rejection membrane 120. Typically, the analyte sensinglayer 110 is an enzyme layer. Most typically, the analyte sensing layer110 comprises an enzyme capable of producing and/or utilizing oxygenand/or hydrogen peroxide, for example the enzyme glucose oxidase.Optionally the enzyme in the analyte sensing layer is combined with asecond carrier protein such as human serum albumin, bovine serum albuminor the like. In an illustrative embodiment, an oxidoreductase enzymesuch as glucose oxidase in the analyte sensing layer 110 reacts withglucose to produce hydrogen peroxide, a compound which then modulates acurrent at an electrode. As this modulation of current depends on theconcentration of hydrogen peroxide, and the concentration of hydrogenperoxide correlates to the concentration of glucose, the concentrationof glucose can be determined by monitoring this modulation in thecurrent. In a specific embodiment of the invention, the hydrogenperoxide is oxidized at a working electrode which is an anode (alsotermed herein the anodic working electrode), with the resulting currentbeing proportional to the hydrogen peroxide concentration. Suchmodulations in the current caused by changing hydrogen peroxideconcentrations can by monitored by any one of a variety of sensordetector apparatuses such as a universal sensor amperometric biosensordetector or one of the other variety of similar devices known in the artsuch as glucose monitoring devices produced by Medtronic MiniMed.

In embodiments of the invention, the analyte sensing layer 110 can beapplied over portions of the conductive layer or over the entire regionof the conductive layer. Typically the analyte sensing layer 110 isdisposed on the working electrode which can be the anode or the cathode.Optionally, the analyte sensing layer 110 is also disposed on a counterand/or reference electrode. While the analyte sensing layer 110 can beup to about 1000 microns (μm) in thickness, typically the analytesensing layer is relatively thin as compared to those found in sensorspreviously described in the art, and is for example, typically less than1, 0.5, 0.25 or 0.1 microns in thickness. As discussed in detail below,some methods for generating a thin analyte sensing layer 110 includebrushing the layer onto a substrate (e.g. the reactive surface of aplatinum black electrode), as well as spin coating processes, dip anddry processes, low shear spraying processes, ink-jet printing processes,silk screen processes and the like.

Typically, the analyte sensing layer 110 is coated and or disposed nextto one or more additional layers. Optionally, the one or more additionallayers includes a protein layer 116 disposed upon the analyte sensinglayer 110. Typically, the protein layer 116 comprises a protein such ashuman serum albumin, bovine serum albumin or the like. Typically, theprotein layer 116 comprises human serum albumin. In some embodiments ofthe invention, an additional layer includes an analyte modulating layer112 that is disposed above the analyte sensing layer 110 to regulateanalyte access with the analyte sensing layer 110. For example, theanalyte modulating membrane layer 112 can comprise a glucose limitingmembrane, which regulates the amount of glucose that contacts an enzymesuch as glucose oxidase that is present in the analyte sensing layer.Such glucose limiting membranes can be made from a wide variety ofmaterials known to be suitable for such purposes, e.g., siliconecompounds such as polydimethyl siloxanes, polyurethanes, polyureacellulose acetates, NAFION, polyester sulfonic acids (e.g. Kodak AQ),hydrogels, the polymer blends disclosed herein or any other suitablehydrophilic membranes known to those skilled in the art.

In some embodiments of the invention, an adhesion promoter layer 114 isdisposed between layers such as the analyte modulating layer 112 and theanalyte sensing layer 110 as shown in FIG. 1 in order to facilitatetheir contact and/or adhesion. In a specific embodiment of theinvention, an adhesion promoter layer 114 is disposed between theanalyte modulating layer 112 and the protein layer 116 as shown in FIG.1 in order to facilitate their contact and/or adhesion. The adhesionpromoter layer 114 can be made from any one of a wide variety ofmaterials known in the art to facilitate the bonding between suchlayers. Typically, the adhesion promoter layer 114 comprises a silanecompound. In alternative embodiments, protein or like molecules in theanalyte sensing layer 110 can be sufficiently crosslinked or otherwiseprepared to allow the analyte modulating membrane layer 112 to bedisposed in direct contact with the analyte sensing layer 110 in theabsence of an adhesion promoter layer 114.

Embodiments of typical elements used to make the sensors disclosedherein are discussed below.

Typical Analyte Sensor Constituents Used in Embodiments of the Invention

The following disclosure provides examples of typicalelements/constituents used in sensor embodiments of the invention. Whilethese elements can be described as discreet units (e.g. layers), thoseof skill in the art understand that sensors can be designed to containelements having a combination of some or all of the material propertiesand/or functions of the elements/constituents discussed below (e.g. anelement that serves both as a supporting base constituent and/or aconductive constituent and/or a matrix for the analyte sensingconstituent and which further functions as an electrode in the sensor).Those in the art understand that these thin film analyte sensors can beadapted for use in a number of sensor systems such as those describedbelow.

Base Constituent

Sensors of the invention typically include a base constituent (see, e.g.element 102 in FIG. 1 ). The term “base constituent” is used hereinaccording to art accepted terminology and refers to the constituent inthe apparatus that typically provides a supporting matrix for theplurality of constituents that are stacked on top of one another andcomprise the functioning sensor. In one form, the base constituentcomprises a thin film sheet of insulative (e.g. electrically insulativeand/or water impermeable) material. This base constituent can be made ofa wide variety of materials having desirable qualities such asdielectric properties, water impermeability and hermeticity. Somematerials include metallic, and/or ceramic and/or polymeric substratesor the like.

The base constituent may be self-supporting or further supported byanother material as is known in the art. In one embodiment of the sensorconfiguration shown in FIG. 1 , the base constituent 102 comprises aceramic. Alternatively, the base constituent comprises a polymericmaterial such as a polyimmide. In an illustrative embodiment, theceramic base comprises a composition that is predominantly Al₂O₃ (e.g.96%). The use of alumina as an insulating base constituent for use withimplantable devices is disclosed in U.S. Pat. Nos. 4,940,858, 4,678,868and 6,472,122 which are incorporated herein by reference. The baseconstituents of the invention can further include other elements knownin the art, for example hermetical vias (see, e.g. WO 03/023388).Depending upon the specific sensor design, the base constituent can berelatively thick constituent (e.g. thicker than 50, 100, 200, 300, 400,500 or 1000 microns). Alternatively, one can utilize a nonconductiveceramic, such as alumina, in thin constituents, e.g., less than about 30microns.

Conductive Constituent

The electrochemical sensors of the invention typically include aconductive constituent disposed upon the base constituent that includesat least one electrode for measuring an analyte or its byproduct (e.g.oxygen and/or hydrogen peroxide) to be assayed (see, e.g. element 104 inFIG. 1 ). The term “conductive constituent” is used herein according toart accepted terminology and refers to electrically conductive sensorelements such as electrodes which are capable of measuring and adetectable signal and conducting this to a detection apparatus. Anillustrative example of this is a conductive constituent that canmeasure an increase or decrease in current in response to exposure to astimuli such as the change in the concentration of an analyte or itsbyproduct as compared to a reference electrode that does not experiencethe change in the concentration of the analyte, a coreactant (e.g.oxygen) used when the analyte interacts with a composition (e.g. theenzyme glucose oxidase) present in analyte sensing constituent 110 or areaction product of this interaction (e.g. hydrogen peroxide).Illustrative examples of such elements include electrodes which arecapable of producing variable detectable signals in the presence ofvariable concentrations of molecules such as hydrogen peroxide oroxygen. Typically one of these electrodes in the conductive constituentis a working electrode, which can be made from non-corroding metal orcarbon. A carbon working electrode may be vitreous or graphitic and canbe made from a solid or a paste. A metallic working electrode may bemade from platinum group metals, including palladium or gold, or anon-corroding metallically conducting oxide, such as ruthenium dioxide.Alternatively, the electrode may comprise a silver/silver chlorideelectrode composition. The working electrode may be a wire or a thinconducting film applied to a substrate, for example, by coating orprinting. Typically, only a portion of the surface of the metallic orcarbon conductor is in electrolytic contact with the analyte-containingsolution. This portion is called the working surface of the electrode.The remaining surface of the electrode is typically isolated from thesolution by an electrically insulating cover constituent 106. Examplesof useful materials for generating this protective cover constituent 106include polymers such as polyimides, polytetrafluoroethylene,polyhexafluoropropylene and silicones such as polysiloxanes.

In addition to the working electrode, the analyte sensors of theinvention typically include a reference electrode or a combinedreference and counter electrode (also termed a quasi-reference electrodeor a counter/reference electrode). If the sensor does not have acounter/reference electrode then it may include a separate counterelectrode, which may be made from the same or different materials as theworking electrode. Typical sensors of the present invention have one ormore working electrodes and one or more counter, reference, and/orcounter/reference electrodes. One embodiment of the sensor of thepresent invention has two, three or four or more working electrodes.These working electrodes in the sensor may be integrally connected orthey may be kept separate.

Typically for in vivo use, embodiments of the present invention areimplanted subcutaneously in the skin of a mammal for direct contact withthe body fluids of the mammal, such as blood. Alternatively, the sensorscan be implanted into other regions within the body of a mammal such asin the intraperotineal space. When multiple working electrodes are used,they may be implanted together or at different positions in the body.The counter, reference, and/or counter/reference electrodes may also beimplanted either proximate to the working electrode(s) or at otherpositions within the body of the mammal. Embodiments of the inventioninclude sensors comprising electrodes constructed from nanostructuredmaterials. As used herein, a “nanostructured material” is an objectmanufactured to have at least one dimension smaller than 100 nm.Examples include, but are not limited to, single-walled nanotubes,double-walled nanotubes, multi-walled nanotubes, bundles of nanotubes,fullerenes, cocoons, nanowires, nanofibres, onions and the like.

Interference Rejection Constituent

The electrochemical sensors of the invention optionally include aninterference rejection constituent disposed between the surface of theelectrode and the environment to be assayed. In particular, certainsensor embodiments rely on the oxidation and/or reduction of hydrogenperoxide generated by enzymatic reactions on the surface of a workingelectrode at a constant potential applied. Because amperometricdetection based on direct oxidation of hydrogen peroxide requires arelatively high oxidation potential, sensors employing this detectionscheme may suffer interference from oxidizable species that are presentin biological fluids such as ascorbic acid, uric acid and acetaminophen.In this context, the term “interference rejection constituent” is usedherein according to art accepted terminology and refers to a coating ormembrane in the sensor that functions to inhibit spurious signalsgenerated by such oxidizable species which interfere with the detectionof the signal generated by the analyte to be sensed. Certaininterference rejection constituents function via size exclusion (e.g. byexcluding interfering species of a specific size). Examples ofinterference rejection constituents include one or more layers orcoatings of compounds such as hydrophilic crosslinked pHEMA andpolylysine polymers as well as cellulose acetate (including celluloseacetate incorporating agents such as poly(ethylene glycol)),polyethersulfones, polytetra-fluoroethylenes, the perfluoronated ionomerNAFION, polyphenylenediamine, epoxy and the like. Illustrativediscussions of such interference rejection constituents are found forexample in Ward et al., Biosensors and Bioelectronics 17 (2002) 181-189and Choi et al., Analytical Chimica Acta 461 (2002) 251-260 which areincorporated herein by reference. Other interference rejectionconstituents include for example those observed to limit the movement ofcompounds based upon a molecular weight range, for example celluloseacetate as disclosed for example in U.S. Pat. No. 5,755,939, thecontents of which are incorporated by reference. Additional compositionshaving an unexpected constellation of material properties that make themideal for use as interference rejection membranes in certainamperometric glucose sensors as well as methods for making and usingthem are disclosed herein, for example in U.S. patent application Ser.No. 12/572,087.

Analyte Sensing Constituent

The electrochemical sensors of the invention include an analyte sensingconstituent disposed on the electrodes of the sensor (see, e.g. element110 in FIG. 1 ). The term “analyte sensing constituent” is used hereinaccording to art accepted terminology and refers to a constituentcomprising a material that is capable of recognizing or reacting with ananalyte whose presence is to be detected by the analyte sensorapparatus. Typically this material in the analyte sensing constituentproduces a detectable signal after interacting with the analyte to besensed, typically via the electrodes of the conductive constituent. Inthis regard the analyte sensing constituent and the electrodes of theconductive constituent work in combination to produce the electricalsignal that is read by an apparatus associated with the analyte sensor.Typically, the analyte sensing constituent comprises an oxidoreductaseenzyme capable of reacting with and/or producing a molecule whose changein concentration can be measured by measuring the change in the currentat an electrode of the conductive constituent (e.g. oxygen and/orhydrogen peroxide), for example the enzyme glucose oxidase. An enzymecapable of producing a molecule such as hydrogen peroxide can bedisposed on the electrodes according to a number of processes known inthe art. The analyte sensing constituent can coat all or a portion ofthe various electrodes of the sensor. In this context, the analytesensing constituent may coat the electrodes to an equivalent degree.Alternatively, the analyte sensing constituent may coat differentelectrodes to different degrees, with for example the coated surface ofthe working electrode being larger than the coated surface of thecounter and/or reference electrode.

Typical sensor embodiments of this element of the invention utilize anenzyme (e.g. glucose oxidase) that has been combined with a secondprotein (e.g. albumin) in a fixed ratio (e.g. one that is typicallyoptimized for glucose oxidase stabilizing properties) and then appliedon the surface of an electrode to form a thin enzyme constituent. In atypical embodiment, the analyte sensing constituent comprises a GOx andHSA mixture. In a typical embodiment of an analyte sensing constituenthaving GOx, the GOx reacts with glucose present in the sensingenvironment (e.g. the body of a mammal) and generates hydrogen peroxideaccording to the reaction shown in FIG. 1 , wherein the hydrogenperoxide so generated is anodically detected at the working electrode inthe conductive constituent.

As noted above, the enzyme and the second protein (e.g. an albumin) aretypically treated to form a crosslinked matrix (e.g. by adding across-linking agent to the protein mixture). As is known in the art,crosslinking conditions may be manipulated to modulate factors such asthe retained biological activity of the enzyme, its mechanical and/oroperational stability. Illustrative crosslinking procedures aredescribed in U.S. patent application Ser. No. 10/335,506 and PCTpublication WO 03/035891 which are incorporated herein by reference. Forexample, an amine cross-linking reagent, such as, but not limited to,glutaraldehyde, can be added to the protein mixture.

Protein Constituent

The electrochemical sensors of the invention optionally include aprotein constituent disposed between the analyte sensing constituent andthe analyte modulating constituent (see, e.g. element 116 in FIG. 1 ).The term “protein constituent” is used herein according to art acceptedterminology and refers to constituent containing a carrier protein orthe like that is selected for compatibility with the analyte sensingconstituent and/or the analyte modulating constituent. In typicalembodiments, the protein constituent comprises an albumin such as humanserum albumin. The HSA concentration may vary between about 0.5%-30%(w/v). Typically the HSA concentration is about 1-10% w/v, and mosttypically is about 5% w/v. In alternative embodiments of the invention,collagen or BSA or other structural proteins used in these contexts canbe used instead of or in addition to HSA. This constituent is typicallycrosslinked on the analyte sensing constituent according to art acceptedprotocols.

Adhesion Promoting Constituent

The electrochemical sensors of the invention can include one or moreadhesion promoting (AP) constituents (see, e.g. element 114 in FIG. 1 ).The term “adhesion promoting constituent” is used herein according toart accepted terminology and refers to a constituent that includesmaterials selected for their ability to promote adhesion betweenadjoining constituents in the sensor. Typically, the adhesion promotingconstituent is disposed between the analyte sensing constituent and theanalyte modulating constituent. Typically, the adhesion promotingconstituent is disposed between the optional protein constituent and theanalyte modulating constituent. The adhesion promoter constituent can bemade from any one of a wide variety of materials known in the art tofacilitate the bonding between such constituents and can be applied byany one of a wide variety of methods known in the art. Typically, theadhesion promoter constituent comprises a silane compound such asγ-aminopropyltrimethoxysilane.

The use of silane coupling reagents, especially those of the formulaR′Si(OR)₃ in which R′ is typically an aliphatic group with a terminalamine and R is a lower alkyl group, to promote adhesion is known in theart (see, e.g. U.S. Pat. No. 5,212,050 which is incorporated herein byreference). For example, chemically modified electrodes in which asilane such as γ-aminopropyltriethoxysilane and glutaraldehyde were usedin a step-wise process to attach and to co-crosslink bovine serumalbumin (BSA) and glucose oxidase (GOx) to the electrode surface arewell known in the art (see, e.g. Yao, T. Analytica Chim. Acta 1983, 148,27-33).

In certain embodiments of the invention, the adhesion promotingconstituent further comprises one or more compounds that can also bepresent in an adjacent constituent such as the polydimethyl siloxane(PDMS) compounds that serves to limit the diffusion of analytes such asglucose through the analyte modulating constituent. In illustrativeembodiments the formulation comprises 0.5-20% PDMS, typically 5-15%PDMS, and most typically 10% PDMS. In certain embodiments of theinvention, the adhesion promoting constituent is crosslinked within thelayered sensor system and correspondingly includes an agent selected forits ability to crosslink a moiety present in a proximal constituent suchas the analyte modulating constituent. In illustrative embodiments ofthe invention, the adhesion promoting constituent includes an agentselected for its ability to crosslink an amine or carboxyl moiety of aprotein present in a proximal constituent such a the analyte sensingconstituent and/or the protein constituent and or a siloxane moietypresent in a compound disposed in a proximal layer such as the analytemodulating layer.

Analyte Modulating Constituent

The electrochemical sensors of the invention include an analytemodulating constituent disposed on the sensor (see, e.g. element 112 inFIG. 1 ). The term “analyte modulating constituent” is used hereinaccording to art accepted terminology and refers to a constituent thattypically forms a membrane on the sensor that operates to modulate thediffusion of one or more analytes, such as glucose, through theconstituent. In certain embodiments of the invention, the analytemodulating constituent is an analyte-limiting membrane (e.g. a glucoselimiting membrane) which operates to prevent or restrict the diffusionof one or more analytes, such as glucose, through the constituents. Inother embodiments of the invention, the analyte-modulating constituentoperates to facilitate the diffusion of one or more analytes, throughthe constituents. Optionally such analyte modulating constituents can beformed to prevent or restrict the diffusion of one type of moleculethrough the constituent (e.g. glucose), while at the same time allowingor even facilitating the diffusion of other types of molecules throughthe constituent (e.g. 02). Typically, the analyte modulating constituentcomprises a polycarbonate polymer composition as disclosed herein.

With respect to glucose sensors, in known enzyme electrodes, glucose andoxygen from blood, as well as some interferents, such as ascorbic acidand uric acid, diffuse through a primary membrane of the sensor. As theglucose, oxygen and interferents reach the analyte sensing constituent,an enzyme, such as glucose oxidase, catalyzes the conversion of glucoseto hydrogen peroxide and gluconolactone. The hydrogen peroxide maydiffuse back through the analyte modulating constituent, or it maydiffuse to an electrode where it can be reacted to form oxygen and aproton to produce a current that is proportional to the glucoseconcentration. The sensor membrane assembly serves several functions,including selectively allowing the passage of glucose therethrough. Inthis context, an illustrative analyte modulating constituent is asemi-permeable membrane which permits passage of water, oxygen and atleast one selective analyte and which has the ability to absorb water,the membrane having a water soluble, hydrophilic polymer.

A variety of illustrative analyte modulating compositions are known inthe art and are described for example in U.S. Pat. Nos. 6,319,540,5,882,494, 5,786,439 5,777,060, 5,771,868 and 5,391,250, the disclosuresof each being incorporated herein by reference. The hydrogels describedtherein are particularly useful with a variety of implantable devicesfor which it is advantageous to provide a surrounding water constituent.In typical embodiments of the invention, the analyte modulatingcomposition includes the polycarbonate polymeric compositions disclosedherein.

Cover Constituent

The electrochemical sensors of the invention include one or more coverconstituents which are typically electrically insulating protectiveconstituents (see, e.g. element 106 in FIG. 1 ). Typically, such coverconstituents can be in the form of a coating, sheath or tube and aredisposed on at least a portion of the analyte modulating constituent.Acceptable polymer coatings for use as the insulating protective coverconstituent can include, but are not limited to, non-toxic biocompatiblepolymers such as silicone compounds, polyimides, biocompatible soldermasks, epoxy acrylate copolymers, or the like. Further, these coatingscan be photo-imageable to facilitate photolithographic forming ofapertures through to the conductive constituent. A typical coverconstituent comprises spun on silicone. As is known in the art, thisconstituent can be a commercially available RTV (room temperaturevulcanized) silicone composition. A typical chemistry in this context ispolydimethyl siloxane (acetoxy based).

Illustrative Embodiments of Analyte Sensor Apparatus and AssociatedCharacteristics

The analyte sensor apparatus disclosed herein has a number ofembodiments. A general embodiment of the invention is an analyte sensorapparatus for implantation within a mammal. While the analyte sensorsare typically designed to be implantable within the body of a mammal,the sensors are not limited to any particular environment and caninstead be used in a wide variety of contexts, for example for theanalysis of most liquid samples including biological fluids such aswhole-blood, lymph, plasma, serum, saliva, urine, stool, perspiration,mucus, tears, cerebrospinal fluid, nasal secretion, cervical or vaginalsecretion, semen, pleural fluid, amniotic fluid, peritoneal fluid,middle ear fluid, joint fluid, gastric aspirate or the like. Inaddition, solid or desiccated samples may be dissolved in an appropriatesolvent to provide a liquid mixture suitable for analysis.

As noted above, the sensor embodiments disclosed herein can be used tosense analytes of interest in one or more physiological environments. Incertain embodiments for example, the sensor can be in direct contactwith interstitial fluids as typically occurs with subcutaneous sensors.The sensors of the present invention may also be part of a skin surfacesystem where interstitial glucose is extracted through the skin andbrought into contact with the sensor (see, e.g. U.S. Pat. Nos. 6,155,992and 6,706,159 which are incorporated herein by reference). In otherembodiments, the sensor can be in contact with blood as typically occursfor example with intravenous sensors. The sensor embodiments of theinvention further include those adapted for use in a variety ofcontexts. In certain embodiments for example, the sensor can be designedfor use in mobile contexts, such as those employed by ambulatory users.Alternatively, the sensor can be designed for use in stationary contextssuch as those adapted for use in clinical settings. Such sensorembodiments include, for example, those used to monitor one or moreanalytes present in one or more physiological environments in ahospitalized patient.

Sensors of the invention can also be incorporated in to a wide varietyof medical systems known in the art. Sensors of the invention can beused, for example, in a closed loop infusion systems designed to controlthe rate that medication is infused into the body of a user. Such aclosed loop infusion system can include a sensor and an associated meterwhich generates an input to a controller which in turn operates adelivery system (e.g. one that calculates a dose to be delivered by amedication infusion pump). In such contexts, the meter associated withthe sensor may also transmit commands to, and be used to remotelycontrol, the delivery system. Typically, the sensor is a subcutaneoussensor in contact with interstitial fluid to monitor the glucoseconcentration in the body of the user, and the liquid infused by thedelivery system into the body of the user includes insulin. Illustrativesystems are disclosed for example in U.S. Pat. Nos. 6,558,351 and6,551,276; PCT Application Nos. US99/21703 and US99/22993; as well as WO2004/008956 and WO 2004/009161, all of which are incorporated herein byreference.

Certain embodiments of the invention measure peroxide and have theadvantageous characteristic of being suited for implantation in avariety of sites in the mammal including regions of subcutaneousimplantation and intravenous implantation as well as implantation into avariety of non-vascular regions. A peroxide sensor design that allowsimplantation into non-vascular regions has advantages over certainsensor apparatus designs that measure oxygen due to the problems withoxygen noise that can occur in oxygen sensors implanted intonon-vascular regions. For example, in such implanted oxygen sensorapparatus designs, oxygen noise at the reference sensor can compromisethe signal to noise ratio which consequently perturbs their ability toobtain stable glucose readings in this environment. The peroxide sensorsof the invention therefore overcome the difficulties observed with suchoxygen sensors in non-vascular regions.

Certain peroxide sensor embodiments of the invention further includeadvantageous long term or “permanent” sensors which are suitable forimplantation in a mammal for a time period of greater than 30 days. Inparticular, as is known in the art (see, e.g. ISO 10993, BiologicalEvaluation of Medical Devices) medical devices such as the sensorsdescribed herein can be categorized into three groups based on implantduration: (1) “Limited” (<24 hours), (2) “Prolonged” (24 hours-30 days),and (3) “Permanent” (>30 days). In some embodiments of the invention,the design of the peroxide sensor of the invention allows for a“Permanent” implantation according to this categorization, i.e. >30days. In related embodiments of the invention, the highly stable designof the peroxide sensor of the invention allows for an implanted sensorto continue to function in this regard for 2, 3, 4, 5, 6 or 12 or moremonths.

Permutations of Analyte Sensor Apparatus and Elements

As noted above, the invention disclosed herein includes a number ofembodiments including sensors having constellations of elementsincluding polycarbonate polymeric membranes. Such embodiments of theinvention allow artisans to generate a variety of permutations of theanalyte sensor apparatus disclosed herein. As noted above, illustrativegeneral embodiments of the sensor disclosed herein include a base layer,a cover layer and at least one layer having a sensor element such as anelectrode disposed between the base and cover layers. Typically, anexposed portion of one or more sensor elements (e.g., a workingelectrode, a counter electrode, reference electrode, etc.) is coatedwith a very thin layer of material having an appropriate electrodechemistry. For example, an enzyme such as lactate oxidase, glucoseoxidase, glucose dehydrogenase or hexokinase, can be disposed on theexposed portion of the sensor element within an opening or aperturedefined in the cover layer. FIG. 1 illustrates a cross-section of atypical sensor structure 100 of the present invention. The sensor isformed from a plurality of layers of various conductive andnon-conductive constituents disposed on each other according to a methodof the invention to produce a sensor structure 100.

As noted above, in the sensors of the invention, the various layers(e.g. the analyte sensing layer) of the sensors can have one or morebioactive and/or inert materials incorporated therein. The term“incorporated” as used herein is meant to describe any state orcondition by which the material incorporated is held on the outersurface of or within a solid phase or supporting matrix of the layer.Thus, the material “incorporated” may, for example, be immobilized,physically entrapped, attached covalently to functional groups of thematrix layer(s). Furthermore, any process, reagents, additives, ormolecular linker agents which promote the “incorporation” of saidmaterial may be employed if these additional steps or agents are notdetrimental to, but are consistent with the objectives of the presentinvention. This definition applies, of course, to any of the embodimentsof the present invention in which a bioactive molecule (e.g. an enzymesuch as glucose oxidase) is “incorporated.” For example, certain layersof the sensors disclosed herein include a proteinaceous substance suchas albumin which serves as a crosslinkable matrix. As used herein, aproteinaceous substance is meant to encompass substances which aregenerally derived from proteins whether the actual substance is a nativeprotein, an inactivated protein, a denatured protein, a hydrolyzedspecies, or a derivatized product thereof. Examples of suitableproteinaceous materials include, but are not limited to enzymes such asglucose oxidase and lactate oxidase and the like, albumins (e.g. humanserum albumin, bovine serum albumin etc.), caseins, gamma-globulins,collagens and collagen derived products (e.g., fish gelatin, fish glue,animal gelatin, and animal glue).

An illustrative embodiment of the invention is shown in FIG. 1 . Thisembodiment includes an electrically insulating base layer 102 to supportthe sensor 100. The electrically insulating layer base 102 can be madeof a material such as a ceramic substrate, which may be self-supportingor further supported by another material as is known in the art. In analternative embodiment, the electrically insulating layer 102 comprisesa polyimide substrate, for example a polyimide tape, dispensed from areel. Providing the layer 102 in this form can facilitate clean, highdensity mass production. Further, in some production processes usingsuch a polyimide tape, sensors 100 can be produced on both sides of thetape.

Typical embodiments of the invention include an analyte sensing layerdisposed on the base layer 102. In an illustrative embodiment as shownin FIG. 1 the analyte sensing layer comprises a conductive layer 104which is disposed on insulating base layer 102. Typically the conductivelayer 104 comprises one or more electrodes. The conductive layer 104 canbe applied using many known techniques and materials as will bedescribed hereafter, however, the electrical circuit of the sensor 100is typically defined by etching the disposed conductive layer 104 into adesired pattern of conductive paths. A typical electrical circuit forthe sensor 100 comprises two or more adjacent conductive paths withregions at a proximal end to form contact pads and regions at a distalend to form sensor electrodes. An electrically insulating protectivecover layer 106 such as a polymer coating is typically disposed onportions of the conductive layer 104. Acceptable polymer coatings foruse as the insulating protective layer 106 can include, but are notlimited to, non-toxic biocompatible polymers such as polyimide,biocompatible solder masks, epoxy acrylate copolymers, or the like.Further, these coatings can be photo-imageable to facilitatephotolithographic forming of apertures 108 through to the conductivelayer 104. In certain embodiments of the invention, an analyte sensinglayer is disposed upon a porous metallic and/or ceramic and/or polymericmatrix with this combination of elements functioning as an electrode inthe sensor.

In the sensors of the present invention, one or more exposed regions orapertures 108 can be made through the protective layer 106 to theconductive layer 104 to define the contact pads and electrodes of thesensor 100. In addition to photolithographic development, the apertures108 can be formed by a number of techniques, including laser ablation,chemical milling or etching or the like. A secondary photoresist canalso be applied to the cover layer 106 to define the regions of theprotective layer to be removed to form the apertures 108. An operatingsensor 100 typically includes a plurality of electrodes such as aworking electrode and a counter electrode electrically isolated fromeach other, however typically situated in close proximity to oneanother. Other embodiments may also include a reference electrode. Stillother embodiments may utilize a separate reference element not formed onthe sensor. The exposed electrodes and/or contact pads can also undergosecondary processing through the apertures 108, such as additionalplating processing, to prepare the surfaces and/or strengthen theconductive regions.

An analyte sensing layer 110 is typically disposed on one or more of theexposed electrodes of the conductive layer 104 through the apertures108. Typically, the analyte sensing layer 110 is a sensor chemistrylayer and most typically an enzyme layer. Typically, the analyte sensinglayer 110 comprises the enzyme glucose oxidase or the enzyme lactateoxidase. In such embodiments, the analyte sensing layer 110 reacts withglucose to produce hydrogen peroxide which modulates a current to theelectrode which can be monitored to measure an amount of glucosepresent. The sensor chemistry layer 110 can be applied over portions ofthe conductive layer or over the entire region of the conductive layer.Typically the sensor chemistry layer 110 is disposed on portions of aworking electrode and a counter electrode that comprise a conductivelayer. Some methods for generating the thin sensor chemistry layer 110include spin coating processes, dip and dry processes, low shearspraying processes, ink-jet printing processes, silk screen processesand the like. Most typically the thin sensor chemistry layer 110 isapplied using a spin coating process.

The analyte sensing layer 110 is typically coated with one or morecoating layers. In some embodiments of the invention, one such coatinglayer includes a membrane which can regulate the amount of analyte thatcan contact an enzyme of the analyte sensing layer. For example, acoating layer can comprise an analyte modulating membrane layer such asa glucose limiting membrane which regulates the amount of glucose thatcontacts the glucose oxidase enzyme layer on an electrode. Such glucoselimiting membranes can be made from a wide variety of materials known tobe suitable for such purposes, e.g., silicone, polyurethane, polyureacellulose acetate, Nafion, polyester sulfonic acid (Kodak AQ), hydrogelsor any other membrane known to those skilled in the art. In certainembodiments of the invention, the analyte modulating layer comprises alinear polyurethane/polyurea polymer polycarbonate with a branchedacrylate hydrophilic comb-copolymer having a central chain and aplurality of side chains coupled to the central chain, wherein at leastone side chain comprises a silicone moiety.

In some embodiments of the invention, a coating layer is a glucoselimiting membrane layer 112 which is disposed above the sensor chemistrylayer 110 to regulate glucose contact with the sensor chemistry layer110. In some embodiments of the invention, an adhesion promoter layer114 is disposed between the membrane layer 112 and the sensor chemistrylayer 110 as shown in FIG. 1 in order to facilitate their contact and/oradhesion. The adhesion promoter layer 114 can be made from any one of awide variety of materials known in the art to facilitate the bondingbetween such layers. Typically, the adhesion promoter layer 114comprises a silane compound. In alternative embodiments, protein or likemolecules in the sensor chemistry layer 110 can be sufficientlycrosslinked or otherwise prepared to allow the membrane layer 112 to bedisposed in direct contact with the sensor chemistry layer 110 in theabsence of an adhesion promoter layer 114.

As noted above, embodiments of the present invention can include one ormore functional coating layers. As used herein, the term “functionalcoating layer” denotes a layer that coats at least a portion of at leastone surface of a sensor, more typically substantially all of a surfaceof the sensor, and that is capable of interacting with one or moreanalytes, such as chemical compounds, cells and fragments thereof, etc.,in the environment in which the sensor is disposed. Non-limitingexamples of functional coating layers include sensor chemistry layers(e.g., enzyme layers), analyte limiting layers, biocompatible layers;layers that increase the slipperiness of the sensor; layers that promotecellular attachment to the sensor; layers that reduce cellularattachment to the sensor; and the like. Typically analyte modulatinglayers operate to prevent or restrict the diffusion of one or moreanalytes, such as glucose, through the layers. Optionally such layerscan be formed to prevent or restrict the diffusion of one type ofmolecule through the layer (e.g. glucose), while at the same timeallowing or even facilitating the diffusion of other types of moleculesthrough the layer (e.g. O₂). An illustrative functional coating layer isa hydrogel such as those disclosed in U.S. Pat. Nos. 5,786,439 and5,391,250, the disclosures of each being incorporated herein byreference. The hydrogels described therein are particularly useful witha variety of implantable devices for which it is advantageous to providea surrounding water layer.

The sensor embodiments disclosed herein can include layers havingUV-absorbing polymers. In accordance with one aspect of the presentinvention, there is provided a sensor including at least one functionalcoating layer including an UV-absorbing polymer. In some embodiments,the UV-absorbing polymer is a polyurethane, a polyurea or apolyurethane/polyurea copolymer. More typically, the selectedUV-absorbing polymer is formed from a reaction mixture including adiisocyanate, at least one diol, diamine or mixture thereof, and apolyfunctional UV-absorbing monomer.

UV-absorbing polymers are used with advantage in a variety of sensorfabrication methods, such as those described in U.S. Pat. No. 5,390,671,to Lord et al., entitled “Transcutaneous Sensor Insertion Set”; U.S.Pat. No. 5,165,407, to Wilson et al., entitled “Implantable GlucoseSensor”; and U.S. Pat. No. 4,890,620, to Gough, entitled“Two-Dimensional Diffusion Glucose Substrate Sensing Electrode”, whichare incorporated herein in their entireties by reference. However, anysensor production method which includes the step of forming anUV-absorbing polymer layer above or below a sensor element is consideredto be within the scope of the present invention. In particular, theinventive methods are not limited to thin-film fabrication methods, andcan work with other sensor fabrication methods that utilize UV-lasercutting. Embodiments can work with thick-film, planar or cylindricalsensors and the like, and other sensor shapes requiring laser cutting.

As disclosed herein, the sensors of the present invention areparticularly designed for use as subcutaneous or transcutaneous glucosesensors for monitoring blood glucose levels in a diabetic patient.Typically each sensor comprises a plurality of sensor elements, forexample electrically conductive elements such as elongated thin filmconductors, formed between an underlying insulative thin film base layerand an overlying insulative thin film cover layer.

If desired, a plurality of different sensor elements can be included ina single sensor. For example, both conductive and reactive sensorelements can be combined in one sensor, optionally with each sensorelement being disposed on a different portion of the base layer. One ormore control elements can also be provided. In such embodiments, thesensor can have defined in its cover layer a plurality of openings orapertures. One or more openings can also be defined in the cover layerdirectly over a portion of the base layer, in order to provide forinteraction of the base layer with one or more analytes in theenvironment in which the sensor is disposed. The base and cover layerscan be comprised of a variety of materials, typically polymers. In morespecific embodiments the base and cover layers are comprised of aninsulative material such as a polyimide. Openings are typically formedin the cover layer to expose distal end electrodes and proximal endcontact pads. In a glucose monitoring application, for example, thesensor can be placed transcutaneously so that the distal end electrodesare in contact with patient blood or extracellular fluid, and thecontact pads are disposed externally for convenient connection to amonitoring device.

Analyte Sensor Apparatus Configurations

In a clinical setting, accurate and relatively fast determinations ofanalytes such as glucose and/or lactate levels can be determined fromblood samples utilizing electrochemical sensors. Conventional sensorsare fabricated to be large, comprising many serviceable parts, or small,planar-type sensors which may be more convenient in many circumstances.The term “planar” as used herein refers to the well-known procedure offabricating a substantially planar structure comprising layers ofrelatively thin materials, for example, using the well-known thick orthin-film techniques. See, for example, Liu et al., U.S. Pat. No.4,571,292, and Papadakis et al., U.S. Pat. No. 4,536,274, both of whichare incorporated herein by reference. As noted below, embodiments of theinvention disclosed herein have a wider range of geometricalconfigurations (e.g. planar) than existing sensors in the art. Inaddition, certain embodiments of the invention include one or more ofthe sensors disclosed herein coupled to another apparatus such as amedication infusion pump.

FIG. 2 provides a diagrammatic view of a typical analyte sensorconfiguration of the current invention. Certain sensor configurationsare of a relatively flat “ribbon” type configuration that can be madewith the analyte sensor apparatus. Such “ribbon” type configurationsillustrate an advantage of the sensors disclosed herein that arises dueto the spin coating of sensing enzymes such as glucose oxidase, amanufacturing step that produces extremely thin enzyme coatings thatallow for the design and production of highly flexible sensorgeometries. Such thin enzyme coated sensors provide further advantagessuch as allowing for a smaller sensor area while maintaining sensorsensitivity, a highly desirable feature for implantable devices (e.g.smaller devices are easier to implant). Consequently, sensor embodimentsof the invention that utilize very thin analyte sensing layers that canbe formed by processes such as spin coating can have a wider range ofgeometrical configurations (e.g. planar) than those sensors that utilizeenzyme layers formed via processes such as electrodeposition.

Certain sensor configurations include multiple conductive elements suchas multiple working, counter and reference electrodes. Advantages ofsuch configurations include increased surface area which provides forgreater sensor sensitivity. For example, one sensor configurationintroduces a third working sensor. One obvious advantage of such aconfiguration is signal averaging of three sensors which increasessensor accuracy. Other advantages include the ability to measuremultiple analytes. In particular, analyte sensor configurations thatinclude electrodes in this arrangement (e.g. multiple working, counterand reference electrodes) can be incorporated into multiple analytesensors. The measurement of multiple analytes such as oxygen, hydrogenperoxide, glucose, lactate, potassium, calcium, and any otherphysiologically relevant substance/analyte provides a number ofadvantages, for example the ability of such sensors to provide a linearresponse as well as ease in calibration and/or recalibration.

An exemplary multiple sensor device comprises a single device having afirst sensor which is polarized cathodically and designed to measure thechanges in oxygen concentration that occur at the working electrode (acathode) as a result of glucose interacting with glucose oxidase; and asecond sensor which is polarized anodically and designed to measurechanges in hydrogen peroxide concentration that occurs at the workingelectrode (an anode) as a result of glucose coming form the externalenvironment and interacting with glucose oxidase. As is known in theart, in such designs, the first oxygen sensor will typically experiencea decrease in current at the working electrode as oxygen contacts thesensor while the second hydrogen peroxide sensor will typicallyexperience an increase in current at the working electrode as thehydrogen peroxide generated as shown in FIG. 1 contacts the sensor. Inaddition, as is known in the art, an observation of the change incurrent that occurs at the working electrodes as compared to thereference electrodes in the respective sensor systems correlates to thechange in concentration of the oxygen and hydrogen peroxide moleculeswhich can then be correlated to the concentration of the glucose in theexternal environment (e.g. the body of the mammal).

The analyte sensors of the invention can be coupled with other medicaldevices such as medication infusion pumps. In an illustrative variationof this scheme, replaceable analyte sensors of the invention can becoupled with other medical devices such as medication infusion pumps,for example by the use of a port couple to the medical device (e.g. asubcutaneous port with a locking electrical connection).

Illustrative Methods and Materials for Making Analyte Sensor Apparatusof the Invention

A number of articles, U.S. patents and patent application describe thestate of the art with the common methods and materials disclosed hereinand further describe various elements (and methods for theirmanufacture) that can be used in the sensor designs disclosed herein.These include for example, U.S. Pat. Nos. 6,413,393; 6,368,274;5,786,439; 5,777,060; 5,391,250; 5,390,671; 5,165,407, 4,890,620,5,390,671, 5,390,691, 5,391,250, 5,482,473, 5,299,571, 5,568,806; UnitedStates Patent Application 20020090738; as well as PCT InternationalPublication Numbers WO 01/58348, WO 03/034902, WO 03/035117, WO03/035891, WO 03/023388, WO 03/022128, WO 03/022352, WO 03/023708, WO03/036255, WO03/036310 and WO 03/074107, the contents of each of whichare incorporated herein by reference.

Typical sensors for monitoring glucose concentration of diabetics arefurther described in Shichiri, et al., “In Vivo Characteristics ofNeedle-Type Glucose Sensor-Measurements of Subcutaneous GlucoseConcentrations in Human Volunteers,” Horm. Metab. Res., Suppl. Ser.20:17-20 (1988); Bruckel, et al.: “In Vivo Measurement of SubcutaneousGlucose Concentrations with an Enzymatic Glucose Sensor and a WickMethod,” Klin. Wochenschr. 67:491-495 (1989); and Pickup, et al.: “InVivo Molecular Sensing in Diabetes Mellitus: An Implantable GlucoseSensor with Direct Electron Transfer,” Diabetologia 32:213-217 (1989).Other sensors are described in, for example Reach, et al., in ADVANCESIN IMPLANTABLE DEVICES, A. Turner (ed.), JAI Press, London, Chap. 1,(1993), incorporated herein by reference.

A typical embodiment of the invention disclosed herein is a method ofmaking a sensor apparatus for implantation within a mammal comprisingthe steps of: providing a base layer; forming a conductive layer on thebase layer, wherein the conductive layer includes an electrode (andtypically a working electrode, a reference electrode and a counterelectrode); forming an analyte sensing layer on the conductive layer,wherein the analyte sensing layer includes a composition that can alterthe electrical current at the electrode in the conductive layer in thepresence of an analyte; optionally forming a protein layer on theanalyte sensing layer; forming an adhesion promoting layer on theanalyte sensing layer or the optional protein layer; forming an analytemodulating layer disposed on the adhesion promoting layer, wherein theanalyte modulating layer includes a composition that modulates thediffusion of the analyte therethrough; and forming a cover layerdisposed on at least a portion of the analyte modulating layer, whereinthe cover layer further includes an aperture over at least a portion ofthe analyte modulating layer. In certain embodiments of the invention,the analyte modulating layer comprises a linear polyurethane/polyureapolymer polycarbonate with a branched acrylate copolymer having acentral chain and a plurality of side chains coupled to the centralchain. In some embodiments of these methods, the analyte sensorapparatus is formed in a planar geometric configuration

As disclosed herein, the various layers of the sensor can bemanufactured to exhibit a variety of different characteristics which canbe manipulated according to the specific design of the sensor. Forexample, the adhesion promoting layer includes a compound selected forits ability to stabilize the overall sensor structure, typically asilane composition. In some embodiments of the invention, the analytesensing layer is formed by a spin coating process and is of a thicknessselected from the group consisting of less than 1, 0.5, 0.25 and 0.1microns in height.

Typically, a method of making the sensor includes the step of forming aprotein layer on the analyte sensing layer, wherein a protein within theprotein layer is an albumin selected from the group consisting of bovineserum albumin and human serum albumin. Typically, a method of making thesensor includes the step of forming an analyte sensing layer thatcomprises an enzyme composition selected from the group consisting ofglucose oxidase, glucose dehydrogenase, lactate oxidase, hexokinase andlactate dehydrogenase. In such methods, the analyte sensing layertypically comprises a carrier protein composition in a substantiallyfixed ratio with the enzyme, and the enzyme and the carrier protein aredistributed in a substantially uniform manner throughout the analytesensing layer.

Electrodes of the invention can be formed from a wide variety ofmaterials known in the art. For example, the electrode may be made of anoble late transition metals. Metals such as gold, platinum, silver,rhodium, iridium, ruthenium, palladium, or osmium can be suitable invarious embodiments of the invention. Other compositions such as carbonor mercury can also be useful in certain sensor embodiments. Of thesemetals, silver, gold, or platinum is typically used as a referenceelectrode metal. A silver electrode which is subsequently chloridized istypically used as the reference electrode. These metals can be depositedby any means known in the art, including the plasma deposition methodcited, supra, or by an electroless method which may involve thedeposition of a metal onto a previously metallized region when thesubstrate is dipped into a solution containing a metal salt and areducing agent. The electroless method proceeds as the reducing agentdonates electrons to the conductive (metallized) surface with theconcomitant reduction of the metal salt at the conductive surface. Theresult is a layer of adsorbed metal. (For additional discussions onelectroless methods, see: Wise, E. M. Palladium: Recovery, Properties,and Uses, Academic Press, New York, N.Y. (1988); Wong, K. et al. Platingand Surface Finishing 1988, 75, 70-76; Matsuoka, M. et al. Ibid. 1988,75, 102-106; and Pearlstein, F. “Electroless Plating,” ModernElectroplating, Lowenheim, F. A., Ed., Wiley, New York, N.Y. (1974),Chapter 31.). Such a metal deposition process must yield a structurewith good metal to metal adhesion and minimal surface contamination,however, to provide a catalytic metal electrode surface with a highdensity of active sites. Such a high density of active sites is aproperty necessary for the efficient redox conversion of anelectroactive species such as hydrogen peroxide.

In an exemplary embodiment of the invention, the base layer is initiallycoated with a thin film conductive layer by electrode deposition,surface sputtering, or other suitable process step. In one embodimentthis conductive layer may be provided as a plurality of thin filmconductive layers, such as an initial chrome-based layer suitable forchemical adhesion to a polyimide base layer followed by subsequentformation of thin film gold-based and chrome-based layers in sequence.In alternative embodiments, other electrode layer conformations ormaterials can be used. The conductive layer is then covered, inaccordance with conventional photolithographic techniques, with aselected photoresist coating, and a contact mask can be applied over thephotoresist coating for suitable photoimaging. The contact masktypically includes one or more conductor trace patterns for appropriateexposure of the photoresist coating, followed by an etch step resultingin a plurality of conductive sensor traces remaining on the base layer.In an illustrative sensor construction designed for use as asubcutaneous glucose sensor, each sensor trace can include threeparallel sensor elements corresponding with three separate electrodessuch as a working electrode, a counter electrode and a referenceelectrode.

Portions of the conductive sensor layers are typically covered by aninsulative cover layer, typically of a material such as a siliconpolymer and/or a polyimide. The insulative cover layer can be applied inany desired manner. In an exemplary procedure, the insulative coverlayer is applied in a liquid layer over the sensor traces, after whichthe substrate is spun to distribute the liquid material as a thin filmoverlying the sensor traces and extending beyond the marginal edges ofthe sensor traces in sealed contact with the base layer. This liquidmaterial can then be subjected to one or more suitable radiation and/orchemical and/or heat curing steps as are known in the art. Inalternative embodiments, the liquid material can be applied using spraytechniques or any other desired means of application. Various insulativelayer materials may be used such as photoimagable epoxyacrylate, with anillustrative material comprising a photoimagable polyimide availablefrom OCG, Inc. of West Paterson, N.J., under the product number 7020.

As noted above, appropriate electrode chemistries defining the distalend electrodes can be applied to the sensor tips, optionally subsequentto exposure of the sensor tips through the openings. In an illustrativesensor embodiment having three electrodes for use as a glucose sensor,an enzyme (typically glucose oxidase) is provided within one of theopenings, thus coating one of the sensor tips to define a workingelectrode. One or both of the other electrodes can be provided with thesame coating as the working electrode. Alternatively, the other twoelectrodes can be provided with other suitable chemistries, such asother enzymes, left uncoated, or provided with chemistries to define areference electrode and a counter electrode for the electrochemicalsensor.

Methods for producing the extremely thin enzyme coatings of theinvention include spin coating processes, dip and dry processes, lowshear spraying processes, ink-jet printing processes, silk screenprocesses and the like. As artisans can readily determine the thicknessof an enzyme coat applied by process of the art, they can readilyidentify those methods capable of generating the extremely thin coatingsof the invention. Typically, such coatings are vapor crosslinkedsubsequent to their application. Surprisingly, sensors produced by theseprocesses have material properties that exceed those of sensors havingcoatings produced by electrodeposition including enhanced longevity,linearity, regularity as well as improved signal to noise ratios. Inaddition, embodiments of the invention that utilize glucose oxidasecoatings formed by such processes are designed to recycle hydrogenperoxide and improve the biocompatibility profiles of such sensors.

Sensors generated by processes such as spin coating processes also avoidother problems associated with electrodeposition, such as thosepertaining to the material stresses placed on the sensor during theelectrodeposition process. In particular, the process ofelectrodeposition is observed to produce mechanical stresses on thesensor, for example mechanical stresses that result from tensile and/orcompression forces. In certain contexts, such mechanical stresses mayresult in sensors having coatings with some tendency to crack ordelaminate. This is not observed in coatings disposed on sensor via spincoating or other low-stress processes. Consequently, yet anotherembodiment of the invention is a method of avoiding theelectrodeposition influenced cracking and/or delamination of a coatingon a sensor comprising applying the coating via a spin coating process.

Methods for Using Analyte Sensor Apparatus of the Invention

A related embodiment of the invention is a method of sensing an analytewithin the body of a mammal, the method comprising implanting an analytesensor embodiment disclosed herein in to the mammal and then sensing analteration in current at the working electrode and correlating thealteration in current with the presence of the analyte, so that theanalyte is sensed. The analyte sensor can polarized anodically such thatthe working electrode where the alteration in current is sensed is ananode, or cathodically such that the working electrode where thealteration in current is sensed is a cathode. In one such method, theanalyte sensor apparatus senses glucose in the mammal. In an alternativemethod, the analyte sensor apparatus senses lactate, potassium, calcium,oxygen, pH, and/or any physiologically relevant analyte in the mammal.

Certain analyte sensors having the structure discussed above have anumber of highly desirable characteristics which allow for a variety ofmethods for sensing analytes in a mammal. For example, in such methods,the analyte sensor apparatus implanted in the mammal functions to sensean analyte within the body of a mammal for more than 1, 2, 3, 4, 5, or 6months. Typically, the analyte sensor apparatus so implanted in themammal senses an alteration in current in response to an analyte within15, 10, 5 or 2 minutes of the analyte contacting the sensor. In suchmethods, the sensors can be implanted into a variety of locations withinthe body of the mammal, for example in both vascular and non-vascularspaces.

EXAMPLES

The following examples are given to aid in understanding the invention,but it is to be understood that the invention is not limited to theparticular materials or procedures of examples. All materials used inthe examples were obtained from commercial sources.

Example 1: Synthesis and Characterization of IllustrativePolyurea/Polyurethane Polymers Using Conventional Methods

The disclosure provided herein in combination with what is known in thatart confirms that functional linear polyurethane/polyurea polymers canbe made from a number of formulations, for example those disclosed inU.S. Pat. Nos. 5,777,060; 5,882,494; 6,642,015; and PCT publications WO96/30431; WO 96/18115; WO 98/13685; and WO 98/17995, the contents ofwhich are incorporated herein by reference. Certain of these polymersprovide formulations useful as a glucose limiting membrane (GLM).

A standard GLM formulation used to make embodiments of the inventioncomprises:

25 mol % polymethylhydrosiloxane (PDMS), trimethylsilyl terminated,25-35 centistokes;

75 mol % polypropylene glycol diamine (Jeffamine 600, apolyoxyalkyleneamine with an approximate molecular weight of 600); and50 mol % of a diisocyanate (e.g., 4,4′-diisocyanate). This standard GLMformulation and processes for its synthesis are disclosed for example inU.S. Pat. Nos. 6,642,015, 5,777,060 and 6,642,015.

Another formulation used in embodiments of the invention is termed a“half permeable GLM”, due to the observation that its glucosepermeability is one-half of the standard formulation immediately above.In the standard GLM, the Jeffamine/PDMS ration=3/1 (mole ratio). Incontrast, in the “half permeable GLM”, this ratio is altered so that theJeffamine/PDMS=12/1. This half-permeable GLM is can be used for exampleto reduce the weight % of GLM-urea in an overall polymer blend in orderto reach a particular Isig (or glucose permeability). Also, the presenceof more GLM-acrylate polymer in the polymer blend can enhance theadhesion between polycarbonate polymeric membrane layer and a proximallayer in a sensor (e.g. one comprising glucose oxidase).

The invention claimed is:
 1. An amperometric analyte sensor comprising: a base layer; a conductive layer disposed on the base layer and comprising a working electrode; an analyte sensing layer disposed on the conductive layer; and an analyte modulating layer disposed on the analyte sensing layer, wherein the analyte modulating layer: (a) is formed by a reaction mixture comprising: from 17% to 23% weight percent hexamethylene diisocyanate; from 0% to 8.5% weight percent methylene diphenyl diisocyanate; from 14% to 48% weight percent polydimethylsiloxane having amino terminal groups; and from 7.5% to 19% weight percent poly(1,6-hexyl carbonate) diol; and a catalyst present in the reaction mixture in amounts less than 0.2% of reaction mixture components; and (b) exhibits a greater thermal stability than a comparable analyte modulating layer formed from a reaction mixture where the catalyst is present in the formulation in amounts greater than or equal to 0.2% of the reaction mixture.
 2. The amperometric analyte sensor of claim 1, wherein the amperometric analyte sensor is a glucose sensor comprising an architecture adapted to measure glucose in interstitial fluids.
 3. The amperometric analyte sensor of claim 1, further comprising at least one of: a protein layer disposed on the analyte sensing layer; or a cover layer disposed on the analyte sensor apparatus, wherein the cover layer comprises an aperture positioned on the cover layer so as to facilitate an analyte present in an in vivo environment from contacting and diffusing through an analyte modulating layer; and contacting the analyte sensing layer.
 4. The amperometric analyte sensor of claim 1, wherein the conductive layer comprises a plurality of electrodes including a working electrode, a counter electrode and a reference electrode.
 5. The amperometric analyte sensor of claim 4, wherein the conductive layer comprises a plurality of working electrodes and/or counter electrodes and/or reference electrodes; and optionally the plurality of working, counter and reference electrodes are grouped together as a unit and positionally distributed on the conductive layer in a repeating pattern of units.
 6. The amperometric analyte sensor of claim 1, wherein the analyte modulating layer is formed by a reaction mixture further comprising a polycarbonate.
 7. The amperometric analyte sensor of claim 1, wherein the catalyst is present in the reaction mixture in amounts less than 0.11% of the reaction mixture.
 8. The amperometric analyte sensor of claim 1, wherein the analyte modulating layer comprises: about 22% hexamethylene diisocyanate; about 3.5% methylene diphenyl diisocyanate; about 22.5% polydimethylsiloxane having amino terminal groups; and about 7.5% poly(1,6-hexyl carbonate) diol.
 9. The amperometric analyte sensor of claim 1, wherein water is included as a chain extender in the reaction mixture.
 10. The amperometric analyte sensor of claim 1, wherein thermal stability of the analyte modulating layer is measured by observing changes in molecular weight of the analyte modulating layer maintained at a temperature of 60° C. over at least 3, 5 or 7 days.
 11. An amperometric analyte sensor comprising: a base layer; a conductive layer disposed on the base layer and comprising a working electrode; an analyte sensing layer disposed on the conductive layer; and an analyte modulating layer disposed on the analyte sensing layer, wherein the analyte modulating layer: (a) is formed by a reaction mixture comprising: from 17% to 23% weight percent hexamethylene diisocyanate; from 0% to 8.5% weight percent methylene diphenyl diisocyanate; from 14% to 48% weight percent polydimethylsiloxane having amino terminal groups; a polycarbonate diol; and a catalyst present in the reaction mixture in amounts less than 0.2% of reaction mixture components; and (b) exhibits a greater thermal stability than a comparable analyte modulating layer formed from a reaction mixture where the catalyst is present in the formulation in amounts greater than or equal to 0.2% of the reaction mixture.
 12. The amperometric analyte sensor of claim 11, wherein the amperometric analyte sensor is a glucose sensor comprising an architecture adapted to measure glucose in interstitial fluids.
 13. The amperometric analyte sensor of claim 11, wherein the catalyst is present in the reaction mixture in amounts less than 0.11% of the reaction mixture. 